Research method for improving cerebral apoplexy neuroinflammation mechanism through vagus nerve electrical stimulation

Abstract

The invention discloses a research method for improving a cerebral apoplexy neuroinflammation mechanism through vagus nerve electrical stimulation, and relates to the field of medicine. Comprising the following steps: constructing an SD rat MCAO / R model by adopting a suture method, and incorporating the model into an experiment according to a Longa-Z score of 1-3; the method comprises the following steps: dividing rats into three groups, namely a false operation SHAM group, a cerebral ischemia reperfusion MCAO / R group and a cerebral ischemia reperfusion vagus nerve electrical stimulation MCAO + VNS group; vagus nerve electrical stimulation VNS intervention: after ischemia is carried out for 30 min, the left vagus nerve is stimulated. VNS-Dectin1 mechanism association is disclosed for the first time, Western blot and immunofluorescence co-localization prove that VNS significantly reduces the protein expression of brain tissue Dectin1 and reduces co-localization of Dectin1 and IBA1 + microglial cells, and it is clear that VNS regulates activation of microglial cells by inhibiting a Dectin1 receptor.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to a research method for improving the mechanism of nerve inflammation in cerebral stroke by vagus nerve electrical stimulation. BACKGROUND

[0002] At present, in the treatment of ischemic stroke, microglia cells gather in the cerebral infarction area and activate the Dectin1 receptor, triggering a nerve inflammation storm and exacerbating brain tissue damage. Although vagus nerve electrical stimulation (VNS) is used in clinical practice to improve the motor function of stroke patients (its application is originally derived from epilepsy treatment), its specific anti-inflammatory mechanism has not been clearly defined.

[0003] In the prior art, the effect of VNS on cerebral stroke is mainly limited to the observation of behavioral improvement (such as motor function score), and there is a lack of in-depth research on molecular targets. In particular, there is no literature report on whether Dectin1 receptor is involved in the anti-inflammatory process of VNS. Traditional anti-inflammatory treatment (such as drug inhibition of inflammatory factors) has problems such as insufficient targeting and significant side effects, and the regulation strategy targeting the specific receptor of microglia cells (such as Dectin1) has not been explored in combination with VNS.

[0004] Therefore, the present application proposes a research method for improving the mechanism of nerve inflammation in cerebral stroke by vagus nerve electrical stimulation. SUMMARY

[0005] The purpose of the present application is to solve the problems existing in the prior art, and to propose a research method for improving the mechanism of nerve inflammation in cerebral stroke by vagus nerve electrical stimulation.

[0006] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0007] The research method for improving the mechanism of nerve inflammation in cerebral stroke by vagus nerve electrical stimulation comprises the following steps:

[0008] S1: An MCAO / R model of SD rats is constructed by using a thread plug method, and rats with a Longa-Z score of 1-3 are included in the experiment;

[0009] S2: The rats are divided into three groups, namely: sham operation SHAM group, cerebral ischemia reperfusion MCAO / R group, and cerebral ischemia reperfusion vagus nerve electrical stimulation MCAO+VNS group;

[0010] S3: Vagus nerve electrical stimulation VNS intervention: stimulating the left vagus nerve after 30 minutes of ischemia;

[0011] S4: Mechanism verification: respectively on rats, neurological function score, motor function detection-rod test, cerebral infarction volume determination-TTC staining, brain tissue Tunnel staining, brain tissue protein extraction and BCA method protein concentration determination, Western blot detection of Dectin1 protein expression.

[0012] Preferably: in the S1 step, the line plug method for constructing the MCAO / R model of SD rats includes the following steps:

[0013] S11: Preoperative preparation: rats are fed for 5 days, fasting for 12 hours before operation, and water freely; the surgical instruments required during the operation are sterilized by high-pressure steam, and the operating table is sterilized by ultraviolet light;

[0014] S12: Anesthesia and fixation: rats are injected with 1% sodium pentobarbital 45mg / kg intraperitoneally, and wait for 5min. The rats are fixed on the operating board in a supine position, and the rats maintain uniform breathing without other actions. The tongue is pulled to one side with forceps to prevent suffocation, and the respiratory tract is kept unobstructed during the operation;

[0015] S13: Tissue separation and embolization, after the rat's neck is shaved, it is disinfected with iodophor. A longitudinal incision is made along the midline of the neck. First, use forceps to separate the right anterior cervical fascia layer layer by layer, expose the anterior cervical muscles, and use a retractor to pull the anterior cervical muscle group to the right side to expose the carotid sheath. Then, slowly and gently separate the left vagus nerve, jugular vein and common carotid artery along the carotid sheath with microforceps. Ligate the common carotid artery at the distal end with 4-0 surgical thread. The common carotid artery is divided into external carotid artery and internal carotid artery after the cross. Ligate the external carotid artery, tie a loose knot at the cross, and temporarily block the blood flow with an artery clamp. ③ Make an oblique incision with a blood vessel scissors between the common carotid artery knot and the cross. Insert the prepared thread plug into the incision through the loose knot to the artery clamp of the internal carotid artery. Tighten the loose knot to fix the thread plug. Loosen the artery clamp and adjust the angle of the thread plug. Continue to insert the thread plug into the intracranial direction until the thread plug mark passes through the cross and the thread plug encounters resistance and cannot continue.

[0016] S14: Reperfusion: place the rat on an electric blanket to maintain body temperature at 37±0.3℃, pull out the thread plug after 90min to restore blood flow, complete reperfusion after vascular occlusion, then suture the cervical tissue layer by layer, and disinfect the suture site with iodophor. After the rats wake up, they are caged and fed freely, and the SHAM group rats are not treated with intracervical thread plug, and the rest of the process is the same as the model group rats.

[0017] Preferably: the S3 step includes the following steps:

[0018] S31: Fix the rat in a supine position, and fully expose the rat's head;

[0019] S32: VNS intervention is performed by vagus nerve electrical stimulation, and the intervention site is the left vagus nerve close to the horizontal neck after a small incision is made on the left ventral neck of the rat close to the midline;

[0020] S33: blunt dissection is performed on the subcutaneous fat, salivary gland, sternum, sternocleidomastoid muscle, and the carotid sheath is incised to expose the vagus nerve;

[0021] S34: a 5mm long segment of the left vagus nerve is isolated and connected to the electrode, which consists of a pair of Teflon-coated silver hooks, and a gauze is used to wipe the low-frequency electrical stimulator to avoid short circuit;

[0022] S35: stimulation is started 30 minutes after the plug is inserted, and the plug is removed after 1 hour of stimulation; the stimulation frequency is 20Hz, the stimulation intensity is 9-15, and the operation mode of the SHAM and MCAO / R groups is the same as that of the MCAO+VNS group except that there is no current, and the remaining steps are the same as those of the stimulation group;

[0023] S36: stimulation is performed by the same researcher at the same time every day for 3 consecutive days.

[0024] Preferably: in the S4 step, the purpose of the neurological function score is to explore the improvement of VNS on the neurological function of the stroke rat.

[0025] Preferably: in the S4 step, the purpose of the motor function detection-rotarod test is to explore the improvement of VNS on the motor function of the stroke rat.

[0026] Preferably: in the S4 step, the purpose of the brain infarction volume determination-TTC staining is to detect the volume of brain infarction in the stroke rat to prove that VNS can reduce the volume of brain infarction, which includes the following steps:

[0027] A1: immediately after the rat is anesthetized, the brain is removed, the surface bloodstains of the brain are washed with pre-cooled PBS solution, the rat brain surface is dried, and then placed in a -20° refrigerator for 10 minutes;

[0028] A2: the frozen brain is removed, five consecutive sections are cut along the coronal plane, each section is about 2mm thick, the sections are placed in TTC solution, 37° incubated for 30 minutes in the dark, and the solution is shaken every 5 minutes and the sections are turned over to ensure that the brain slices are in full contact with the TTC solution for reaction;

[0029] A3: after the reaction is completed, the sections are placed in 4% paraformaldehyde for fixation, stored in the dark, and finally the brain slices are arranged in order for photography and preservation, and the brain infarction volume of each rat is calculated using Image J.

[0030] Preferably: the purpose of the brain tissue Tunnel staining in the S4 step is to detect the cell death of the rat brain, which proves that VNS can save the cell death of the rat brain after stroke, and specifically includes the following steps:

[0031] B1: Brain slice preparation: After the rat is anesthetized, heart perfusion is performed, a needle is inserted into the left ventricle after the heart is exposed, the needle is fixed, the right auricle is cut, physiological saline is injected through the needle until the liver is completely white, then the physiological saline is replaced with 4% paraformaldehyde and slowly injected at a constant speed, until the rat's limbs and tail are stiff, finally the complete rat brain tissue is carefully taken out, fixed in 4% paraformaldehyde for 24 hours, after 24 hours, the residual paraformaldehyde in the brain tissue is washed out with PBS, the surface moisture is wiped dry, and then immersed in 30% sucrose for dehydration until the brain tissue sinks to the bottom, the dehydrated brain tissue is taken out, the surface moisture is wiped dry, and then embedded with OTC embedding agent, placed in a -20°C refrigerator for freezing, and finally sliced on a sectioning machine, the cut brain slices are stored in a 24-well plate containing antifreeze, and stored at -20°C.

[0032] B2: Tunnel staining: the desired slice in the antifreeze is taken out and placed in a new 24-well plate, PBS solution is added for washing 3 times, 5 min each time, proteinase K solution is added to the well plate, 37°C incubation for 25 min, then PBS solution is added for washing 3 times, 5 min each time, 0.3% Triton X-100 solution is added, and the well plate is incubated at room temperature for 20 min, PBS solution is added for washing 3 times, 5 min each time, Tunnel reaction solution is mixed uniformly and added to the well plate to completely cover the brain slices, 37°C incubation for 1 hour, then PBS solution is added for washing 3 times, 5 min each time, finally DAPI is added, incubated at room temperature for 15 min, and then washed with PBS solution, as before, add anti-fluorescence quencher to the slice, and seal the slice with a confocal microscope.

[0033] Preferably: the brain tissue protein extraction and BCA method protein concentration determination in the S4 step include the following steps:

[0034] C1: Brain tissue protein extraction, the extracted brain cortex tissue is transferred to a 1.5ml EP tube, PIRA: protease inhibitor: phosphatase inhibitor = 50: 1: 1 tissue protein lysate is added, the tissue is lysed with ultrasonic waves, and then the tissue is ground with a grinder, and then placed on ice for 15 minutes, then placed in a high-speed low-temperature centrifuge, centrifuged at 12000 rpm, 4°C, 20 min, the supernatant is aspirated with a sterile gun head into a labeled EP tube, and the volume of the extracted supernatant is recorded;

[0035] C2: BCA method protein concentration determination, 0, 1, 2, 4, 8, 12, 16, 20 μl of 0.5 mg / ml protein standard was sequentially added in 96-well plate, then PBS was added to make up to 20 μl per well, forming a concentration gradient of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg / ml, each concentration was made in triplicate; after 50-fold dilution of the sample, 20 μl of each sample was added, each sample was made in triplicate; 200 μl of BCA working solution was added to each well, and it was placed at 37℃ for 20-30 minutes; the absorbance at 547 nm wavelength was determined by using the enzyme label instrument; the standard curve was calculated according to the absorbance and concentration of the standard, and then the protein concentration of the sample was calculated according to the standard curve and the sample absorbance and was balanced; after balancing, 5x loading buffer solution was added in a ratio of 4:1 according to the final volume, mixed uniformly, and placed in a 95℃ metal bath for 10 min until the protein was completely denatured, then after cooling, 200 μl was aliquoted per tube and stored in a-80℃ refrigerator.

[0036] Preferably: in the S4 step, the Western blot detection of Dectin1 protein expression comprises the following steps:

[0037] D1: gel preparation: according to the instruction manual of the Biyun Tian PAGE gel rapid preparation kit, 2.5 ml of A liquid and B liquid for each gel, 50 μl of accelerator was added, mixed uniformly, and quickly added to the glass plate on the gel preparation frame, anhydrous ethanol was added to the upper edge of the short plate, and it was placed for 20 min, then the anhydrous ethanol was poured off, and 0.75 ml of A liquid and B liquid for each plate and 12 μl of accelerator were added, and a ten-hole comb was inserted, ensuring that no bubbles were generated in the gel during the whole process;

[0038] D2: preparation:

[0039] Preparation of electrophoresis solution: weigh glycine 14.4 g, Tris-base 3.03 g, SDS 1.0 g, and mix in L of double-distilled water, mix thoroughly;

[0040] Preparation of transfer solution: weigh glycine 14.4 g, Tris-base 3.03 g, methanol 200 mL, mix in 800 mL of double-distilled water, mix thoroughly;

[0041] Preparation of washing solution TBST: add TBST dry powder buffer (Sevier item number: G0001-2L) to 2 L of double-distilled water, mix thoroughly;

[0042] Electrophoresis: put the sample in the 95℃ metal bath for 5 min in advance, during which the prepared gel is fixed on the electrophoresis frame and placed in the electrophoresis tank, new prepared electrophoresis solution is added to the inner tank to submerge the glass plate, and the tightness of the inner tank is ensured to be good without leakage, the outer tank is added with electrophoresis solution to the scale line position, then the comb is removed, the air bubbles in the channel are removed, 2ul of Marker and 20-30ug of protein are added to the channel, and electrophoresis is carried out at 80V constant voltage until the buffer reaches the bottom;

[0043] D3: Transferring film: the prepared electrotransfer liquid is pre-cooled, the PVDF film of appropriate size is cut, and the methanol solution is activated for 20s in advance, then it is placed in the electrotransfer liquid, and from top to bottom, the black clamp plate-sponge 1 layer-filter paper 3 pieces-gel-PVDF film-filter paper 3 pieces-sponge 1 layer-white clamp plate are sequentially arranged, air bubbles are removed, the clamp plate is clamped tightly, and then it is transferred to the electrotransfer tank, the ice box is placed in the tank, the tank is filled with pre-cooled electrotransfer liquid, and the whole electrotransfer tank is placed in an ice bath, and the film transfer is carried out at a constant current of 250mA, and the film transfer time is calculated according to the target molecule;

[0044] D4: Blocking: 5% skimmed milk is prepared with TBST half an hour in advance and placed on a shaker, after electrotransfer, the PVDF film is transferred to the skimmed milk, and the blocking is carried out at room temperature on the shaker at low speed for 1h, and special target proteins are blocked with Yezhen 1X fast blocking solution for 0.5h;

[0045] D5: Incubating first antibody: after blocking, the solution is washed with TBST for 3 times, 10min each time, then the prepared first antibody solution is added, the first antibody is ensured to cover the strip, and the solution is placed in a 4℃ refrigerator shaker overnight, the concentration of the first antibody is Dectin1 (1:1000), iNOS (1:1000), IL-6 (1:1000), and beta-actin (1:5000);

[0046] D6: Incubating second antibody: the first antibody is recovered, the strip is washed with TBST on the shaker for 5 times, 6min each time, the prepared second antibody solution (1:5000) is added, and the solution is incubated at room temperature on the shaker for 1h;

[0047] D7: Developing: the second antibody is removed, the solution is washed with TBST on the shaker for 5 times, 6min each time, the ECL developer is prepared in the dark, and the Bio-Rad gel imager is used for developing.

[0048] The beneficial effects of the application are:

[0049] 1. The application first discloses the VNS-Dectin1 mechanism correlation, which is verified by Western blot and immunofluorescence co-localization, and VNS significantly reduces the Dectin1 protein expression in brain tissue and reduces the Dectin1 and IBA1+ Co-localization of microglia, which confirmed that VNS regulated microglia activation by inhibiting Dectin1 receptor.

[0050] 2. The present application provides a multi-dimensional therapeutic effect verification system. The behavior level: mNSS score (including motor / sensation / reflex / balance beam test) and rotarod test (latency, maximum speed) quantify the improvement of neural function; the histology level: TTC staining shows that the VNS group has reduced cerebral infarction volume, and TUNEL staining confirms that cell death is reduced; the molecular level: ELISA detects the decrease of inflammatory factors (IL-6, TNF-α) and the increase of IL-10, and Western blot verifies the down-regulation of iNOS and IL-6 protein. BRIEF DESCRIPTION OF DRAWINGS

[0051] Figure 1 The present application provides a research method flow chart for improving cerebral stroke neuroinflammation mechanism by vagus nerve electrical stimulation. DETAILED DESCRIPTION

[0052] The technical solutions of the present application will be further described in detail below in combination with specific embodiments.

[0053] In the description of the present application, it should be noted that unless otherwise explicitly specified and limited, the terms "mounting", "connection", "connection", "setting" should be understood broadly, for example, it can be fixedly connected, set, or detachably connected, set, or integrally connected, set. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0054] Embodiment:

[0055] Experimental animals

[0056] Healthy adult male Sprague-Dawley (SD) rats, weighing 220-260 g, were purchased from the Experimental Animal Center of Chongqing Medical University and were bred by them. At most 5 rats were fed in each cage. All experimental animals were exposed to a 12-hour / 12-hour light and dark cycle, and the indoor temperature was maintained at about 22℃, and the relative humidity was about 50%. The animals to be tested can be free to eat and drink. All animal experimental procedures involved in this study have been approved by the Animal Ethics Committee of Chongqing Medical University.

[0057] Experimental grouping

[0058] The experimental animals were randomly divided into ① sham operation group: SHAM group; ② cerebral ischemia-reperfusion group: MCAO / R group; ③ vagus nerve electrical stimulation after cerebral ischemia-reperfusion group: VNS group, 5 rats in each group.

[0059] Construction of rat MCAO / R model

[0060] According to the previous experimental method, the left cerebral ischemia reperfusion model of SD rats was constructed by thread embolism method. (1) Preoperative preparation: the rats were adaptively fed for 5 days, and the rats were fasted for 12 hours before operation. The surgical instruments needed during the operation were sterilized by high pressure steam, and the operating table was sterilized by ultraviolet rays.

[0061] (2) Anesthesia and fixation: the rats were injected with 1% sodium pentobarbital (45 mg / kg) intraperitoneally, and waited for about 5 min. The rats were fixed on the operating board in a supine position. The rats kept uniform breathing without other movements. The tongue was pulled to one side with forceps to prevent asphyxia, and the respiratory tract was kept unobstructed during the operation.

[0062] (3) Tissue separation and embolization process: ① After the rat's neck was shaved, it was disinfected with iodophor. A longitudinal incision was made along the midline of the neck. First, the right anterior cervical fascia layer was separated layer by layer with forceps, and the anterior cervical muscles were exposed. The anterior cervical muscle group was pulled to the right side with a retractor to fully expose the carotid sheath. ② Along the carotid sheath, the left vagus nerve, jugular vein and common carotid artery were slowly and gently separated with microforceps, and the common carotid artery was ligated at the distal end with 4-0 surgical thread. The common carotid artery was divided into external carotid artery and internal carotid artery after the cross was reached in the head direction. The external carotid artery was ligated, and a loose knot was tied at the cross. The internal carotid artery was temporarily blocked with an artery clamp. ③ A diagonal incision was made between the common carotid artery knot and the cross with a blood vessel scissors. The prepared thread embolism was inserted from the incision through the loose knot to the internal carotid artery clamp. The loose knot was tightened to fix the thread embolism. The artery clamp was loosened, and the angle of the thread embolism was adjusted. The thread embolism was inserted into the intracranial until the thread embolism marker passed through the cross and the thread embolism encountered resistance and could not continue.

[0063] (4) Reperfusion: the rats were placed on an electric blanket to maintain body temperature at 37±0.3℃. After 90 min, the thread embolism was pulled out to restore blood flow. After the blood vessel obstruction, reperfusion was performed. Then the cervical tissues were sutured layer by layer, and the suture site was disinfected with iodophor. After the rats woke up, they were fed in separate cages, and they could eat and drink freely. The rats in the sham group were not treated with internal thread embolism, and the rest of the process was the same as that of the model group rats.

[0064] (5) After the rats woke up after modeling, they were judged according to the Longa-Z score method. If the score was 1-3, they were included in the experiment. The specific standards are as follows: 0 points: no neurological deficit symptoms; 1 point: mild neurological deficit symptoms, lift the rat's tail left forelimb flexion; 2 points: moderate focal neurological deficit symptoms, rats walking to the left side or turning; 3 points: moderate to severe focal neurological deficit symptoms, rats walking to the left side and falling down, unable to walk; 4 points: severe neurological deficit symptoms, decreased level of consciousness, unable to perform spontaneous activities.

[0065] Vagus nerve electrical stimulation intervention

[0066] Rats were fixed in supine position, ensuring the complete exposure of the rat's head. VNS intervention was performed using vagus nerve electrical stimulation, with the intervention site being the left vagus nerve at the level of the neck after making a small incision on the left side of the rat's neck near the midline. The subcutaneous fat, salivary glands, sternum, sternocleidomastoid muscle were blunt dissected, and the carotid sheath was incised to expose the vagus nerve. A 5-mm-long segment of the left vagus nerve was isolated and connected to the electrode. The electrode consisted of a pair of Teflon-coated silver hooks, and to avoid short circuits, the low-frequency stimulator was wiped dry with gauze. Stimulation was started 30 min after the plug was inserted, and the plug was removed after 1 h of stimulation; the stimulation frequency was 20 Hz, and the stimulation intensity was 9-15. The sham operation group and the MCAO group were operated in the same way as the VNS group, except that there was no current, and the rest of the steps were the same as the stimulation group. The stimulation was performed by the same researcher at the same time every day for 3 consecutive days. Neurological function score to explore the improvement effect of VNS on the neurological function of stroke rats

[0067] The neurological function of rats in each group was evaluated using the mNSS scale on the 3rd day after MCAO. The mNSS test consists of several individual tests that assess different neurological functions, including motor function, sensory function, and reflexes. Briefly, the rats were placed on a flat surface and their ability to move their forelimbs and hindlimbs was examined as an indicator of motor function. The score was divided into 0-6 points, with 0 representing normal motor function and 6 representing no movement. Next, the rats' forelimbs and hindlimbs were touched with a soft brush, and their response was examined as an indicator of sensory function. The rats' score was 0-2 points, with 0 representing normal sensory function and 2 representing no response. Finally, the rats' forelimbs and hindlimbs were tapped and their response was examined as an indicator of reflexes. The rats' score was 0-2 points, with 0 representing normal reflexes and 2 representing no response. Then the scores of each test were added up to get the total mNSS score of each rat. The higher the score, the greater the neurological deficit, and the lower the score, the better the neurological function. The specific scores are as follows: Table 1 Modified Neurological Severity Score (mNSS)

[0068]

[0069]

[0070] Motor function test - rotarod experiment to explore the improvement effect of VNS on the motor function of stroke rats

[0071] 3 days before surgery: 2 times a day (interval ≥ 4 hours), gradually accelerated from rest to 10 rpm, single longest 5 minutes;

[0072] Exclusion criteria: those who still cannot maintain balance for > 1 minute after training are excluded.

[0073] 1.2 Official test animal placement: Gently place the rat on the rotarod, ensuring all four limbs are in contact with the rod surface, and simultaneously start the timer and rotation speed;

[0074] Termination criteria:

[0075] Fall (triggering the infrared sensor) or voluntary embrace of the rotarod > 10 seconds; record latency (seconds) and maximum tolerated rotation speed (rpm);

[0076] Repeat design: Each group is tested 3 times (30 minutes apart), and the average is taken.

[0077] 1.3 Behavioral recording video analysis: High-speed camera (120 fps) recording, DeepLabCut 3.0 quantification of gait symmetry;

[0078] Abnormal behavior: Events such as turning around, running in reverse, etc. are recorded by two observers (Kappa > 0.85) in a blind manner.

[0079] Measurement of cerebral infarction volume - TTC staining detects the volume of cerebral infarction in stroke rats to prove that VNS can reduce the volume of cerebral infarction

[0080] Immediately after the rat is euthanized, the brain is removed and the surface bloodstains are rinsed with pre-cooled PBS solution. After the rat's brain surface is dried, it is placed in a -20°C freezer for 10 minutes. After the frozen brain is removed, five consecutive sections are cut along the coronal plane, each with a thickness of about 2mm. The slices are placed in TTC solution, incubated at 37°C for 30 minutes in the dark, and gently shaken every 5 minutes to ensure full contact between the brain slices and the TTC solution. After the reaction is complete, the slices are fixed in 4% paraformaldehyde and stored in the dark. Finally, the brain slices are arranged in order and photographed for preservation. The cerebral infarction volume of each rat is calculated using Image J.

[0081] Brain tissue Tunnel staining detects rat brain cell death to prove that VNS can save stroke rats from brain tissue cell death

[0082] (1) Brain slice preparation: After the rat is anesthetized, cardiac perfusion is performed. After the heart is exposed, a needle is inserted into the left ventricle and fixed. The right auricle is cut, and physiological saline is injected through the needle until the liver is completely white. Then, the physiological saline is replaced with 4% paraformaldehyde, which is slowly and evenly injected until the rat's limbs and tail are stiff. Finally, the intact rat brain tissue is carefully removed and fixed in 4% paraformaldehyde for 24 hours. After 24 hours, the residual paraformaldehyde in the brain tissue is washed out with PBS, and the surface moisture is dried before being immersed in 30% sucrose for dehydration until the brain tissue sinks. After the dehydration of the brain tissue is completed, the surface moisture is dried, and the brain tissue is embedded with OTC embedding agent, then placed in a -20°C freezer. Finally, the slices are cut on a microtome and stored in a 24-well plate containing antifreeze, and stored at -20°C.

[0083] (2) Tunnel staining: The slices needed in the antifreezing solution were placed in a new 24-well plate, and washed with PBS solution for 3 times, 5 min each time. Proteinase K solution was added into the well plate, and incubated at 37 °C for 25 min. Then, the slices were washed with PBS solution for 3 times, 5 min each time. 0.3% Triton X-100 solution was added, and the slices were incubated at room temperature for 20 min, and then washed with PBS solution for 3 times, 5 min each time. Tunnel reaction solution was mixed uniformly, and then added into the well plate to completely cover the slices. After incubation at 37 °C for 1 h, the slices were washed with PBS solution for 3 times, 5 min each time. Finally, DAPI was added, and the slices were incubated at room temperature for 15 min, and then washed with PBS solution. After mounting, the slices were photographed by a confocal microscope.

[0084] Extraction of brain tissue protein

[0085] The extracted brain cortex tissue was transferred into a 1.5 ml EP tube, and the prepared tissue protein lysis solution (PIRA: protease inhibitor: phosphatase inhibitor = 50: 1: 1) was added. The tissue was lysed by ultrasonic, and then ground by a grinder. The tissue was taken out and placed on ice for 15 min, and then placed in a high-speed low-temperature centrifuge. Centrifugation was performed at 12000 rpm, 4 °C, and 20 min. After centrifugation, the supernatant of the tissue was absorbed into a labeled EP tube by a sterile gun head. The volume of the extracted supernatant was recorded.

[0086] BCA method for protein concentration determination

[0087] (1) 0, 1, 2, 4, 8, 12, 16, and 20 μl of 0.5 mg / ml protein standard were sequentially added into a 96-well plate, and then PBS was added to make up to 20 μl per well, forming a concentration gradient of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / ml. Each concentration was made in triplicate.

[0088] (2) After dilution of the sample by 50 times, 20 μl of each sample was added, and each sample was made in triplicate.

[0089] (3) 200 μl of BCA working solution (A solution: B solution = 50: 1) was added to each well, and placed at 37 °C for 20-30 min.

[0090] (4) The absorbance at 547 nm was determined by an enzyme marker.

[0091] (5) The standard curve was calculated according to the absorbance and concentration of the standard, and the protein concentration of the sample was calculated according to the standard curve and the absorbance of the sample, and then balanced.

[0092] (6) After balancing, add 5x Loading Buffer buffer to the last volume at a ratio of 4:1, mix evenly, and place in a 95°C metal bath for 10 min until the protein is completely denatured. After cooling, divide 200 μl per tube and store in a -80°C refrigerator.

[0093] Western blotting detection of dectin1 and inflammation related proteins proves that VNS can inhibit dectin1 receptor and improve cerebral inflammation in stroke rats

[0094] (1) Gel preparation: according to the instructions of the Biyun Tian PAGE gel preparation kit, 2.5 ml of A and B solutions for each gel, add 50 μl of accelerator, mix well, and quickly add to the glass plate on the gel preparation frame, add anhydrous ethanol to the upper edge of the short plate. Let stand for 20 min, pour off the anhydrous ethanol, and add 0.75 ml of A and B solutions for each plate and 12 μl of accelerator, and insert the ten-hole comb. Ensure that no bubbles are formed in the gel during the entire process.

[0095] (2) Preparation of electrophoresis solution: weigh glycine 14.4 g, Tris-base 3.03 g, SDS 1.0 g, and mix in L of double distilled water, mix well.

[0096] Preparation of transfer solution: weigh glycine 14.4 g, Tris-base 3.03 g, methanol 200 mL, mix in 800 mL of double distilled water, and mix well.

[0097] Preparation of washing solution TBST: add TBST dry powder buffer (Sevier catalog number: G0001-2L) to 2 L of double distilled water and mix well.

[0098] Sample electrophoresis: place the sample in a 95°C metal bath for 5 min in advance, during which the prepared gel is fixed on the electrophoresis frame and placed in the electrophoresis tank, add the newly prepared electrophoresis solution to the inner tank to submerge the glass plate, and ensure that the inner tank is tightly sealed and does not leak, and add the electrophoresis solution to the outer tank to the scale line position. Then remove the comb, remove the bubbles in the lane, and add 2 μl of Marker and 20-30 μg of protein in the hole. 80V constant voltage electrophoresis until the buffer reaches the bottom.

[0099] (3) Transfer: The prepared electrotransfer solution was pre-cooled, and the PVDF membrane of appropriate size was cut and activated with methanol solution for 20 s in advance, and then placed in the electrotransfer solution. From top to bottom, the black clamp-sponge 1 layer-3 filter paper-gel-PVDF membrane-3 filter paper-sponge 1 layer-white clamp was arranged in order to exclude air bubbles, and after clamping the clamp, it was transferred to the electrotransfer tank, and an ice box was placed in the tank, and the pre-cooled electrotransfer solution was poured. The whole electrotransfer tank was placed in an ice bath, and electrotransfer was carried out at a constant current of 250 mA. The transfer time was calculated according to the target molecule.

[0100] (4) Blocking: Half an hour in advance, 5% skimmed milk was prepared with TBST and placed on a shaker. After electrotransfer, the PVDF membrane was transferred to the skimmed milk, and the blocking was carried out at room temperature on the shaker for 1 h. Special target proteins were blocked with Yase 1X fast blocking solution for 0.5 h.

[0101] (5) Incubation of primary antibody: After blocking, TBST solution was used for washing 3 times, each for 10 min, and then the prepared primary antibody solution was added to ensure that the primary antibody could cover the band, and it was placed in the 4°C refrigerator shaker overnight. The concentration of primary antibody: Dectin 1 (1:1000), iNOS (1:1000), IL-6 (1:1000), β-actin (1:5000).

[0102] (6) Incubation of secondary antibody: The primary antibody was recovered, and the band was washed with TBST solution on the shaker for 5 times, 6 min / time. The prepared secondary antibody solution (1:5000) was added, and it was incubated at room temperature on the shaker for 1 h.

[0103] (7) Development: The secondary antibody was removed, and TBST solution was used for washing on the shaker for 5 times, 6 min / time. ECL developer was prepared in the dark, and Bio-Rad gel imaging instrument was used for development. The co-localization change of Dectin 1 and microglial cells in brain tissue immunofluorescence staining detection of stroke rats proved that VNS could inhibit the activation of microglial cells induced by Dectin 1 receptor

[0104] 1.1 Specimen preparation

[0105] Deeply anesthetized rats were fixed on the operating board in supine position. The chest cavity and heart were fully exposed by cutting along the right rib, the lower edge of the sternum, and the left rib. The needle was inserted into the ascending aorta from the apex of the heart and clamped with a hemostat to fix the needle. The needle tube was quickly inserted and saline was injected at a constant speed. The right auricle was cut to facilitate blood flow. When the liver was fully whitened, it indicated that the blood had been fully replaced. Then 4% paraformaldehyde was injected. At the beginning of perfusion, the rat's limbs twitched violently. After successful perfusion, the rat's viscera swelled, the limbs stiffened, and the tail stood up in a straight line. Then the head was quickly cut off to take the brain. The skull was cut along the midline with surgical scissors and the brain tissue was removed with tweezers. The brain tissue was soaked in paraformaldehyde for 24 hours, then dehydrated in 10%, 20%, and 30% sucrose solutions. After PBS cleaning, OCT embedding was performed. Freezing sections with a thickness of about 8 μm were cut in the cortical area using a freezing microtome and placed on slides. The slides were baked in a 65°C oven for 1 hour and then stored in a -80°C freezer for later use.

[0106] 1.2 Experimental steps

[0107] (1) The frozen section was taken out of the -80°C freezer and warmed at room temperature for 30 minutes. PBS shaker washing was performed 3 times (5 min / time).

[0108] (2) The liquid around the section was carefully wiped off with filter paper. Triton X-100 was added dropwise and incubated at room temperature for 30 minutes for permeation. PBS shaker washing was performed 3 times (5 min / time).

[0109] (3) A beaker was added with an appropriate amount of sodium citrate buffer to immerse the section. High fire was used for 5 minutes until the liquid boiled, then medium-low fire was used for 10 minutes to complete the microwave antigen repair. The section was taken out and cooled to room temperature.

[0110] (4) PBS shaker washing was performed 3 times (5 min / time). The liquid around the section was carefully wiped off, and the brain tissue position was circled with an immunohistochemical pen. Goat serum was added dropwise and incubated at 37°C for 1 hour.

[0111] (5) The serum on the surface of the brain tissue was spun dry. The section was placed in a wet box, and the diluted primary antibody was added dropwise and incubated at 4°C overnight. Primary antibody (dilution ratio): Dectin1 (1:200), IBA1 (1:200).

[0112] (6) PBS shaker washing was performed 3 times (5 min / time). The diluted fluorescent secondary antibody was added dropwise and incubated at 37°C for 1 hour in the dark.

[0113] (7) PBS shaker washing was performed 3 times (5 min / time). The liquid around the section was carefully wiped off, DAPI Fluoromount-G was added dropwise, a cover glass was placed on top, and the section was incubated at room temperature for 1 hour in the dark after completion of the mounting.

[0114] (8) Use fluorescence microscope to take pictures and observe the co-localization of Dectin1 and cortical microglia and the expression changes.

[0115] Elisa kit-inflammation factor detection to detect the expression of inflammatory factors in stroke rats to prove that VNS can improve nerve inflammation

[0116] 1. Experimental animals and sample processing

[0117] 1.1 After anesthesia, pre-cooled PBS (4℃, 100mL / min) was perfused through the left ventricle to remove blood;

[0118] 1.2 Quickly take the target brain area (such as hippocampus, cortex), and store in liquid nitrogen and-80℃ (≤6 months).

[0119] 2. Main reagents and instruments

[0120] 2.1 Reagent configuration

[0121] Lysis buffer: RIPA (50mM Tris-HCl, pH 7.4, 150mM NaCl, 1% NP-40, 0.5% deoxycholic acid sodium) containing 1× protease inhibitor cocktail (Roche, Cat#04693132001);

[0122] ELISA kit: Commercial detection kit (TNF-α, IL-6, IL-10), standard curve range 15.6-1,000pg / mL.

[0123] 2.2 Instrument parameters

[0124] Microplate reader: BioTek Synergy H1, detection wavelength 450nm / 570nm (reference wavelength);

[0125] Centrifuge: Eppendorf 5425R, 4℃ centrifugation condition 15,000×g 15 minutes.

[0126] 3. Experimental steps

[0127] 3.1 Tissue lysis and protein extraction

[0128] 1. Homogenization: brain tissue (100mg) and lysis solution (1mL) were crushed on ice (20kHz, 10s×3 times, interval 30s ice bath);

[0129] 2. Centrifugation: 15,000×g 4℃ centrifugation for 15 minutes, take supernatant;

[0130] 3. Protein quantification: BCA method (Pierce TMCat#23225), standard curve fitting was done using a quadratic polynomial regression (R 2 >0.99).

[0131] 3.2 ELISA detection procedure

[0132] 1. Coating: 96-well plates were coated with capture antibody (2 μg / mL, 100 μL / well) at 4°C overnight;

[0133] 2. Blocking: Plates were blocked with 5% skim milk (diluted in PBS) for 2 hours at room temperature;

[0134] 3. Incubation:

[0135] Standard / sample (100 μL / well) were detected in duplicate, and incubated for 1 hour at 37°C;

[0136] Biotinylated detection antibody (1:500) was incubated for 45 minutes at 37°C;

[0137] 4. Color development: TMB substrate was incubated for 10 minutes in the dark, and stopped with 2M H2SO4;

[0138] 5. Reading: Dual-wavelength detection (450 nm signal value - 570 nm background value).

[0139] The above description is merely preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art, according to the technical range disclosed by the present application and the inventive concept, can make equivalent replacements or changes, which should be covered in the protection scope of the present application.

Claims

1. A research method for improving the mechanism of neuroinflammation in stroke by vagus nerve electrical stimulation, characterized in that: The following steps are involved: S1: The MCAO / R model was established in SD rats using the suture embolization method, and rats with a Longa-Z score of 1-3 were included in the experiment; S2: The rats were divided into three groups: sham operation SHAM group, cerebral ischemia-reperfusion MCAO / R group, and vagus nerve stimulation MCAO+VNS group after cerebral ischemia-reperfusion; S3: Vagus nerve stimulation (VNS) intervention: stimulation of the left vagus nerve 30 minutes after ischemia; S4: Mechanism verification: The rats were subjected to neurological function scoring, motor function test-rotarod test, cerebral infarction volume measurement-TTC staining, brain tissue tunnel staining, brain tissue protein extraction and BCA method protein concentration determination, and Western blot detection of Dectin1 protein expression.

2. The research method of improving the mechanism of stroke neuroinflammation by vagus nerve electrical stimulation according to claim 1, characterized in that: In step S1, the establishment of the SD rat MCAO / R model by the suture embolization method includes the following steps: S11: Preoperative preparation: Rats were fed for 5 days, fasted for 12 hours before surgery, and had free access to water; S12: Anesthesia and fixation: Rats were intraperitoneally injected with 45 mg / kg of 1% sodium pentobarbital. After 5 minutes, the rats were fixed supine on a surgical splint and their tongues were pulled to one side with forceps to prevent suffocation. S13: Tissue separation and embolization. After depilating the rat's neck and disinfecting it with iodine, a longitudinal incision was made along the midline of the neck. Forceps were used to bluntly separate the right anterior cervical fascia layer by layer to expose the anterior cervical muscles. A retractor was used to pull the anterior cervical muscles to the right to expose the carotid sheath. Microforceps were then used to separate the left vagus nerve, jugular vein, and common carotid artery along the carotid sheath. The common carotid artery was ligated with 4-0 surgical suture at the distal end. An oblique incision was made between the common carotid artery knot and the cervical cross knot with vascular scissors. The prepared suture was inserted from the incision through the slipknot to the internal carotid artery clamp. The knot was tightened, the artery clamp was released, the angle of the suture was adjusted, and the suture was continued to be inserted into the skull until the suture mark passed through the cervical cross and the suture could not be inserted further. S14: Reperfusion: The rats were placed on an electric blanket to maintain their body temperature at 37±0.3℃. After 90 minutes, the suture was removed to restore blood flow and complete reperfusion after vascular occlusion. The neck tissue was then sutured layer by layer, and the suture site was disinfected with iodine. After the rats woke up, they were housed in separate cages and allowed to eat and drink freely. The rats in the SHAM group were not treated with suture insertion in the neck, and the rest of the procedures were the same as those in the model group.

3. The research method of improving the mechanism of stroke neuroinflammation by vagus nerve electrical stimulation according to claim 1, characterized in that: The S3 step comprises the following steps: S31: The rat was fixed in the supine position and the head was completely exposed; S32: VNS intervention was performed using vagus nerve electrical stimulation. The intervention site was the left vagus nerve near the level of the neck after making a small incision on the left ventral side of the neck near the midline of the rat; S33: Blunt dissection of the subcutaneous fat, salivary glands, sternum, and sternocleidomastoid muscle is performed, and the carotid sheath is incised to expose the vagus nerve; S34: A 5-mm segment of the left vagus nerve was isolated and connected to the electrodes; S35: Stimulation started 30 minutes after plug insertion, and the plug was removed after 1 hour of stimulation. The stimulation frequency was 20 Hz, and the stimulation intensity was 9-15. The operation method of the SHAM and MCAO / R groups was the same as that of the MCAO+VNS group, except that no current was applied. The rest of the steps were the same as those of the stimulation group. S36: Stimulation was performed by the same researcher at the same time every day for 3 consecutive days.

4. The research method of improving the mechanism of stroke neuroinflammation by vagus nerve electrical stimulation according to claim 1, characterized in that: In the S4 step, the purpose of the neurological function score is to explore the improvement effect of VNS on the neurological function of stroke rats.

5. The research method of improving the mechanism of stroke neuroinflammation by vagus nerve electrical stimulation according to claim 1, characterized in that: In the S4 step, the purpose of the motor function test-rotarod test is to explore the improvement effect of VNS on the motor function of stroke rats.

6. The research method of improving the mechanism of neuroinflammation in stroke by vagus nerve electrical stimulation according to claim 1, characterized in that: In step S4, the purpose of cerebral infarction volume determination-TTC staining is to detect the volume of cerebral infarction in stroke rats to prove that VNS can reduce cerebral infarction volume, which includes the following steps: A1: After the rats were anesthetized, the brains were immediately removed and the blood stains on the surface of the brains were rinsed with pre-chilled PBS solution. The surface moisture of the brains was wiped dry and the brains were placed in a -20° freezer for 10 min. A2: Remove the frozen brain and cut five consecutive sections along the coronal plane, each section approximately 2 mm thick. Place the sections in TTC solution and incubate at 37°C in the dark for 30 minutes. Shake the solution and flip the sections every 5 minutes to ensure full contact and reaction between the brain slice and the TTC solution. A3: After the reaction is completed, the slices are fixed in 4% paraformaldehyde and stored in the dark. Finally, the brain slices are arranged in order and photographed for storage. Image J is used to calculate the cerebral infarction volume of each rat.

7. The research method of improving the mechanism of stroke neuroinflammation by vagus nerve electrical stimulation according to claim 1, characterized in that: In step S4, the purpose of brain tissue tunnel staining is to detect the death of rat brain cells and prove that VNS can save brain tissue cell death in stroke rats, which specifically includes the following steps: B1: Brain slice preparation: After anesthesia, rats were perfused with the heart. After exposing the heart, a needle was inserted into the left ventricle and fixed. The right atrial appendage was cut and normal saline was injected through the needle until the liver turned completely white. The normal saline was then replaced with 4% paraformaldehyde and injected slowly and evenly until the rat's limbs and tail were stiff. Finally, the intact rat brain tissue was carefully removed and fixed in 4% paraformaldehyde for 24 hours. After 24 hours, the residual paraformaldehyde in the brain tissue was rinsed with PBS to remove the residual paraformaldehyde in the brain tissue. After wiping off the surface moisture, the brain tissue was immersed in 30% sucrose for dehydration until the brain tissue sank to the bottom. The dehydrated brain tissue was removed, the surface moisture was wiped off, and the brain tissue was embedded in OTC embedding medium and placed in a -20° refrigerator for freezing. Finally, the slices were sliced ​​on a microtome and the cut brain slices were stored in a 24-well plate filled with antifreeze at -20°. B2: Tunnel staining: Remove the required slices from the antifreeze solution and place them in a new 24-well plate. Add PBS solution and wash them 3 times, 5 minutes each time. Add proteinase K solution to the well plate and incubate at 37° for 25 minutes. Then wash them 3 times, 5 minutes each time with PBS solution. Add 0.3% Triton X-100 solution and wait for 20 minutes at room temperature. Wash them 3 times, 5 minutes each time with PBS solution. Mix the tunnel reaction solution evenly and add it to the well plate to completely cover the brain slices. Incubate at 37° for 1 hour and then wash them 3 times, 5 minutes each time with PBS solution. Finally, add DAPI and incubate them at room temperature for 15 minutes. Then continue washing with PBS solution. As before, add anti-fluorescence quencher after mounting. After sealing, take pictures with a confocal microscope.

8. The research method of improving the mechanism of stroke neuroinflammation by vagus nerve electrical stimulation according to claim 1, characterized in that: In step S4, the extraction of brain tissue protein and the determination of protein concentration by the BCA method include the following steps: C1: Brain tissue protein extraction: Transfer the extracted cerebral cortex tissue to a 1.5 ml EP tube, add tissue protein lysis buffer with a ratio of PIRA: protease inhibitor: phosphatase inhibitor = 50:1:1, lyse the tissue with ultrasound, and then grind the tissue thoroughly with a grinder. Remove the tissue and let it rest on ice for 15 minutes. Then, place it in a high-speed low-temperature centrifuge and centrifuge it at 12,000 rpm, 4°C, for 20 minutes. After centrifugation, use a sterile pipette tip to aspirate the tissue supernatant into a labeled EP tube, and record the volume of the extracted supernatant. C2: BCA method protein concentration determination, 0, 1, 2, 4, 8, 12, 16, 20 μl of 0.5 mg / ml protein standard was added to a 96-well plate in sequence, and then PBS was added to each well to 20 μl, forming a concentration gradient of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg / ml, and 3 replicates were made for each concentration; after the sample was diluted 50 times, 20 μl was added to each sample, and 3 replicates were made for each sample; 200 μl of BCA working solution was added to each well and incubated at 37°C for 20-30 minutes; the absorbance at a wavelength of 547 nm was measured using a microplate reader; a standard curve was calculated based on the absorbance and concentration of the standard, and the protein concentration of the sample was calculated based on the standard curve and the sample absorbance and balanced; after balancing, 5× Loading Buffer was added at a ratio of 4:1 according to the final volume. Buffer, mix well and place in a 95℃ metal bath for 10 min until the protein is completely denatured. After cooling, dispense 200μl into each tube and store in a -80℃ refrigerator.

9. The research method of improving the mechanism of stroke neuroinflammation by vagus nerve electrical stimulation according to claim 1, characterized in that: In step S4, detecting Dectin1 protein expression by Western blot includes the following steps: D1: Glue preparation: According to the instructions of the Biyuntian PAGE rapid gel preparation kit, add 2.5ml of each lower gel solution A and solution B to each gel, add 50μl of coagulant, mix well, and quickly add to the glass plate on the gel preparation rack. Add anhydrous ethanol to the upper edge of the short plate, let it stand for 20 minutes, pour off the anhydrous ethanol, add 0.75ml of each upper gel solution A and solution B and 12μl of coagulant to each plate, and insert a ten-hole comb; D2: Preparation: Preparation of electrophoresis buffer: Weigh 14.4 g of glycine, 3.03 g of Tris-base, and 1.0 g of SDS, mix in 1 L of double-distilled water, and mix thoroughly. Preparation of transfer buffer: Weigh 14.4 g of glycine, 3.03 g of Tris-base, and 200 mL of methanol, mix in 800 mL of double-distilled water, and mix thoroughly. Preparation of TBST washing solution: Add TBST dry powder buffer to 2L of double-distilled water and mix thoroughly; Sample loading and electrophoresis: Place the sample in a 95°C metal bath for 5 minutes in advance. During this time, fix the prepared gel to the electrophoresis stand and place it in the electrophoresis tank. Add freshly prepared electrophoresis buffer to the inner tank until the glass plate is submerged. Add electrophoresis buffer to the outer tank to the scale line. Then remove the comb and remove any bubbles in the lane. Add 2μl of marker and 20-30μg of protein to the wells. Run electrophoresis at a constant voltage of 80V until the buffer reaches the bottom. D3: Transfer: Pre-cool the prepared electrotransfer solution, cut the PVDF membrane, and activate it with methanol solution for 20 seconds in advance. Then place it in the electrotransfer solution and arrange it in the order of black plywood - 1 layer of sponge - 3 sheets of filter paper - gel - PVDF membrane - 3 sheets of filter paper - 1 layer of sponge - white plywood from top to bottom. Remove bubbles, clamp the plywood, and transfer it to the electrotransfer tank. Place an ice box in the tank and fill it with pre-cooled electrotransfer solution. Place the entire electrotransfer tank in an ice bath and transfer the membrane at a constant current of 250mA. The transfer time is calculated according to the target molecule. D4: Blocking: Prepare 5% skim milk with TBST half an hour in advance and shake on a shaker. After electroporation, transfer the PVDF membrane to skim milk and block it on a shaker at low speed at room temperature for 1 hour. For special target proteins, block with Yazyme 1X rapid blocking solution for 0.5 hour. D5: Incubate with primary antibody: After blocking, wash with TBST solution three times, 10 minutes each time, then add the prepared primary antibody solution to ensure that the primary antibody can cover the bands, and place on a shaker in a 4°C refrigerator overnight; D6: Incubation with secondary antibody: Recover the primary antibody, wash the strips 5 times with TBST solution on a shaker for 6 minutes each time, add the prepared secondary antibody solution, and incubate on a shaker at room temperature for 1 hour; D7: Development: Remove the secondary antibody, wash with TBST solution for 5 times with rapid shaking on a shaker, 6 min / time, prepare ECL developer in the dark, and develop with a Bio-Rad gel imager.

Images