A method for isolating and purifying muscle satellite cells of Shanbei white cashmere goats

Abstract

The application discloses a kind of northern Shaanxi white cashmere goat muscle satellite cell separation and purification method, the present application uses northern Shaanxi white cashmere goat fetus dorsal longest muscle tissue as test material, by aseptic pretreatment, tissue block inversion adherent culture, primary subculture culture Combination of compound technology of hierarchical differential adherent purification, stably obtain the 5th generation muscle satellite cell.The present application optimizes and adapts culture system and purification process, overcomes the defects of low cell yield, serious interference of mixed cells, activity and poor purity of traditional preparation method.At the same time, the present application also adopts RT-PCR molecular identification and immunofluorescence staining combined with multi-level verification method, from gene and protein level Bidirectional verification cell specificity and differentiation potential, accurately determine the biological properties of cell.The present application process is simple, good stability and repeatability, can efficiently prepare northern Shaanxi white cashmere goat muscle satellite cell, provides important technical support for cashmere goat germplasm resource conservation, muscle development mechanism and muscle regeneration related research.

Description

Technical Field

[0001] This invention belongs to the field of animal cell biology technology, specifically relating to a method for isolating and purifying muscle satellite cells of Shaanbei white cashmere goats. Background Technology

[0002] The Shaanbei White Cashmere Goat is a unique and superior local livestock and poultry breed in my country, suitable for both cashmere and meat production. Muscle growth and development capacity is a core breeding trait determining its meat production performance and evaluating its germplasm utilization potential. Muscle satellite cells, as key stem cells mediating skeletal muscle proliferation, damage repair, and tissue regeneration, are crucial in vitro experimental materials for elucidating the regulatory mechanisms of livestock and poultry muscle development and conducting genetic improvement of meat quality traits.

[0003] Currently, there is a significant gap in the technology for isolating, purifying, and identifying fetal muscle satellite cells from Shaanbei white cashmere goats. A standardized and reproducible preparation process suitable for the cell biological characteristics of this breed has not yet been developed in this field. Existing conventional isolation methods generally employ single-enzyme digestion, which easily causes both mechanical and biochemical damage to cells, resulting in decreased cell activity and low survival rates. Furthermore, this process struggles to adequately remove contaminating cells such as fibroblasts, leading to a highly mixed cell population, low purity of target cells, poor experimental reproducibility, and an inability to consistently obtain muscle satellite cells with uniform traits and excellent biological condition. This severely hinders the research progress in elucidating the muscle development mechanism of Shaanbei white cashmere goats, exploring germplasm resources, and promoting innovative industrial applications.

[0004] The longissimus dorsi muscle tissue of the fetus of Shaanbei white cashmere goat used in this invention was obtained from a regular breeding farm in China. The genetic material of this animal is a national-level local animal genetic resource. The sample collection, cell separation and all experimental operations complied with the relevant provisions of the "Animal Husbandry Law of the People's Republic of China" and the "Approval Measures for the Import, Export and Foreign Cooperation Research and Utilization of Animal Genetic Resources". The genetic resource acquisition method was legal and compliant.

[0005] To address the aforementioned deficiencies in existing technologies, this invention establishes an integrated technical system for the isolation, purification, culture, and specific identification of fetal muscle satellite cells adapted to Shaanbei white cashmere goats. This system solves the technical problems of high cell damage, low target cell purity, and poor experimental stability in traditional processes, providing a stable and reliable technical means for the protection of Shaanbei white cashmere goat germplasm resources, basic research on muscle development mechanisms, and genetic improvement of livestock and poultry meat quality traits. Summary of the Invention

[0006] In view of the problems existing in the background art, the purpose of this invention is to provide a method for isolating and purifying muscle satellite cells of Shaanbei white cashmere goats.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A method for isolating and purifying high-purity muscle satellite cells from Shaanbei white cashmere goats, comprising the following steps: S1. Sample aseptic pretreatment: Under aseptic conditions, the longissimus dorsi muscle tissue of fetuses of Shaanbei white cashmere goats aged 110-130 days was selected. The tissue surface was disinfected with ethanol and then thoroughly rinsed with sterile PBS containing double antibodies. S2. Tissue block inverted adherence culture: The pretreated muscle tissue is cut into uniform mince and spread evenly at the bottom of the culture dish. The culture dish is inverted and incubated for 3-5 hours. After the culture dish is upright, complete culture medium is added for constant temperature culture. S3. Primary cell passage culture: When the rate of cell migration from the edge of the tissue block reaches 35% to 45%, remove the residual tissue debris and change the complete culture medium regularly; when the cell confluence reaches 75% to 85%, wash with PBS, digest with trypsin, stop digestion with serum-containing medium, centrifuge, and then resuspend for passage. S4. Differential Adhesion Purification: When the confluence of primary cells reaches 65%–75%, the cells are digested with trypsin at room temperature, digestion is terminated with complete culture medium, and the cells are collected by centrifugation. After resuspending in fresh culture medium, the cells are passaged at a 1:1 ratio and allowed to adhere at room temperature for 25–35 minutes. The difference in adhesion rate between fibroblasts and muscle satellite cells is used to remove contaminating cells. After differential adhesion purification culture, muscle satellite cells of Shaanbei white cashmere goat are obtained.

[0008] Preferably, in step S1, the bispecific antibody is a penicillin-streptomycin mixture with a concentration of 1%.

[0009] Preferably, in step S2, the complete culture medium is prepared by mixing DMEM / F12 culture medium, fetal bovine serum and penicillin-streptomycin mixture.

[0010] Preferably, in step S2, the temperature of the isothermal incubation is 37°C.

[0011] Preferably, in step S3, the complete culture medium is replaced every 1 to 3 days.

[0012] Preferably, in step S4, the specific steps of the graded differential adhesion purification are as follows: each generation up to the 4th generation uses single adhesion purification, and in the 5th generation, the suspended cells are collected and transferred to a new sterile culture dish. The collection and purification are repeated 3 times at 25-35 min intervals.

[0013] Preferably, the method further includes a step of RT-PCR molecular identification of the obtained muscle satellite cells: when the cell confluence reaches 55%–65%, total RNA is extracted from the cells using the TRIzol method, and the target genes are amplified by RT-PCR and detected by electrophoresis; the target genes include muscle differentiation genes, stem cell pluripotency genes, and proliferation-related genes; wherein the muscle differentiation genes are… Myog , Myod1 , Desmin Stem cell pluripotency gene is Klf4 , Sox2 , Nanog The proliferation-related genes are Pcna .

[0014] Preferably, the reaction conditions for the RT-PCR amplification are as follows: pre-denaturation at 95℃ for 5 min; 35 amplification cycles are set, with single cycle parameters as follows: denaturation at 94℃ for 30 s, annealing at 55~60℃ for 30 s, extension at 72℃ for 30 s; after the cycle, final extension at 72℃ for 10 min, and storage at 4℃.

[0015] Preferably, the method further includes an immunofluorescence identification step of the obtained muscle satellite cells; the immunofluorescence identification operation includes: seeding and culturing the purified muscle satellite cells, followed by fixation, permeabilization, blocking, and antibody incubation to complete cell identification; wherein the cell seeding and culture conditions are: 3 × 10⁻⁶ cells / year. 4 Cells were seeded at a density of 1 cell per well in 24-well plates and cultured until the cell confluence reached 75%–85%. The steps for cell fixation, permeabilization and blocking were as follows: cells were washed with sterile PBS, fixed in 4% tissue cell fixative at room temperature, permeabilized in 0.1% Triton X-100 solution, and blocked in 5% BSA blocking solution at room temperature.

[0016] Preferably, in the immunofluorescence identification, the antibody incubation conditions are as follows: Pax7, MyoD1, and MYH3 primary antibodies are incubated overnight at 4°C, and the fluorescent secondary antibody is incubated at room temperature in the dark for 60 minutes.

[0017] Beneficial effects (1) This invention uses an inverted tissue block adherence culture method to isolate longissimus dorsi muscle satellite cells from fetuses of Shaanbei white cashmere goats. It abandons the traditional single-enzyme digestion process, effectively avoiding mechanical and biochemical damage to cells caused by enzymatic digestion. The constructed cell culture system has high stability and excellent reproducibility. This invention can achieve rapid cell adhesion and proliferation. Cells can stably adhere after 12 hours of culture and enter the stage of large-scale rapid proliferation after 1 day of culture. The obtained cells have regular morphology, exhibiting typical morphological characteristics of muscle satellite cells such as spindle shape and inverted triangle shape. The overall biological state of the cells is good, effectively improving the isolation and culture efficiency and survival quality of primary muscle satellite cells from Shaanbei white cashmere goats.

[0018] (2) This invention employs a staged, multi-stage differential adhesion purification process, utilizing the difference in adhesion rates between fibroblasts and muscle satellite cells to enrich target cells in stages and gradually remove contaminating cells, effectively reducing the risk of interference and contamination from contaminating cells. Compared to the traditional single-stage differential adhesion purification process, this invention can significantly improve the purity and bioactivity of target cells, maintain stable cell proliferation characteristics, and effectively improve the technical defects of existing technologies, such as low cell purity, high proportion of contaminating cells, uneven cell growth state, and poor reproducibility.

[0019] (3) This invention employs a multi-level verification method combining RT-PCR molecular identification and immunofluorescence staining to achieve systematic biological function verification of purified muscle satellite cells. This invention uses RT-PCR technology to detect key genes related to cell differentiation, stem cell stemness, and cell proliferation, and combines this with immunofluorescence staining to detect stemness markers and key functional proteins in muscle development, thus verifying the cell's biological characteristics from multiple dimensions. Experimental results show that the cells prepared in this invention can stably express target functional genes and muscle cell-specific characteristic proteins, possess stem cell stemness characteristics and directional muscle differentiation potential. The identification system is complete, the detection results are accurate, and the biological attributes and functional potential of the cells can be clearly defined.

[0020] (4) This invention fills the technological gap in the efficient isolation, purification, standardized culture, and systematic identification of muscle satellite cells from Shaanbei white cashmere goats, and establishes a stable, efficient, and reproducible integrated cell preparation technology system. This invention can provide high-quality, stable in vitro cell models and experimental materials for basic scientific research such as the analysis of the muscle growth and development mechanism and the verification of muscle cell function in Shaanbei white cashmere goats, improve the research and utilization system of Shaanbei white cashmere goat germplasm resources, provide technical support for the genetic improvement of meat quality traits in livestock and poultry, and has good application prospects. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The images are: (a) Morphology of muscle satellite cells cultured for 12 hours after adhesion; (b) Morphology of muscle satellite cells cultured for 1 day after proliferation. Figure 2 The images are: (a) Morphological diagram of muscle satellite cells with 60% confluence; (b) Electrophoresis results of RT-PCR gene identification. Figure 3 This image shows the results of immunofluorescence identification of muscle satellite cell-specific proteins. Detailed Implementation

[0023] In the following description, specific details such as particular system architectures and techniques are set forth for illustrative purposes and not for limitation, in order to provide a thorough understanding of the embodiments of the invention. However, those skilled in the art will understand that the invention may be implemented in other embodiments without these specific details.

[0024] Example 1 A method for isolating and purifying muscle satellite cells from Shaanbei white cashmere goats, the specific steps of which are as follows: S1. Sample Aseptic Pretreatment: Under aseptic conditions, fetuses of Shaanbei white cashmere goats at 120 days gestation were selected, and their longissimus dorsi muscle tissue was rapidly collected. The tissue surface was quickly disinfected once with 75% alcohol to remove adhering bacteria. Subsequently, the tissue was repeatedly washed three times with sterile PBS buffer containing 1% penicillin and streptomycin to thoroughly remove blood, impurities, and residual bacteria from the tissue surface, obtaining sterile and clean muscle tissue samples for later use.

[0025] S2. Tissue Block Inverted Adherent Culture: Using sterile scissors, the pre-treated muscle tissue was thoroughly minced into a uniform paste, which was then evenly spread on the bottom of the cell culture dish. The culture dish was then inverted and placed in a 37°C incubator for 4 hours to ensure the tissue blocks adhered tightly to the bottom. After incubation, the culture dish was inverted, and the prepared complete culture medium was slowly added dropwise. The complete culture medium was prepared by mixing DMEM / F12 basal medium, fetal bovine serum, and penicillin-streptomycin solution in a volume ratio of 79:20:1.

[0026] S3. Primary cell passage culture: During the constant temperature and routine culture period, the cell emergence and growth status were observed daily using an inverted microscope. When the proportion of cells emerging from the edge of the tissue block reached 40%, the residual mushy tissue residue in the culture system was gently removed to prevent impurities from inhibiting cell proliferation. Subsequently, the fresh complete culture medium was replaced every 2 days, and the culture was continued until the cell confluence reached 80%. The cells were thoroughly washed with sterile PBS, digested with trypsin at room temperature for 5 min, and digestion was terminated by adding serum-containing complete culture medium. After centrifugation at 3000 r / min for 5 min, the cells were collected, the supernatant was discarded, and the cell pellet was resuspended in fresh complete culture medium to complete the primary cell passage culture.

[0027] S4. Differential Adhesion Purification: Utilizing the fast adhesion rate of fibroblasts and the slow adhesion rate of muscle satellite cells, contaminating fibroblasts and other cells in the system are removed. Once primary cells reach 70% confluence, they are digested with trypsin at room temperature for 5 minutes, digestion is terminated by adding complete culture medium, and cells are collected by centrifugation at 3000 rpm for 5 minutes. Cells are resuspended in fresh culture medium and passaged at a 1:1 ratio, then allowed to adhere at room temperature for 30 minutes. Up to the 4th generation, purification is achieved through a single static adhesion process per generation. At the 5th generation, the supernatant and suspended cells are collected from the culture dish and transferred to a new sterile culture dish. Suspension cells are collected every 30 minutes, and this purification process is repeated three times to thoroughly remove contaminating cells and improve cell purity. After differential adhesion purification, cells are continuously cultured up to the 5th generation, ultimately yielding stable, high-purity muscle satellite cells from Shaanbei white cashmere goats.

[0028] 1. Cell morphology observation of muscle satellite cells from Shaanbei white cashmere goats was performed, and the results are as follows: Figure 1 As shown.

[0029] Depend on Figure 1 The results showed that the muscle satellite cells of Shaanbei white cashmere goats isolated by this invention were inverted triangles with needle-like ends, exhibiting typical morphological characteristics. After purification, the muscle satellite cells could stably adhere to the culture wall after about 12 hours of culture, with good cell adhesion and no large number of floating or apoptotic cells. After continuous culture for 1 day, the cells entered a rapid proliferation state, and the overall growth morphology changed to a uniform spindle shape, with excellent cell viability and stable growth.

[0030] 2. Molecular identification of muscle satellite cells Gene expression was identified by RT-PCR: when the confluence of purified muscle satellite cells reached 60% (morphological results observed under an inverted microscope are shown in the figure),... Figure 2 (a) Total RNA was extracted from cells using the TRIzol method, and cell-specific gene expression was detected by RT-PCR, covering myoblast differentiation genes, stem cell pluripotency genes, and proliferation-related genes to verify cell biological characteristics. The detected genes included myoblast differentiation genes (…). Myog , Myod1 , Desmin ), stem cell pluripotency gene ( Klf4 , Sox2, Nanog ), proliferation-related genes ( Pcna The primer sequences, accession numbers, and fragment sizes for each gene are shown in Table 1. Table 1. RT-PCR primer sequence information

[0031] The RT-PCR amplification reaction program was set as follows: 95℃ pre-denaturation for 5 min; 35 cycles were performed, specifically: 94℃ denaturation for 30 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s; after the cycles, a final extension at 72℃ for 10 min was performed, and the product was stored at 4℃. The amplified products were detected by 1.5% agarose gel electrophoresis, and the results are as follows. Figure 2 As shown in (b).

[0032] Depend on Figure 2 (a) The results showed that the isolated and cultured muscle satellite cells exhibited typical spindle, fusiform or long spindle shapes, with clear cell boundaries, good adherence and growth, uniform distribution, and no obvious contamination or mass death, which is consistent with the in vitro culture morphological characteristics of muscle satellite cells.

[0033] Depend on Figure 2 (b) The results show that myofascial differentiation-related genes ( Myog , Myod1 , Desmin ), stem cell pluripotency-related genes ( Klf4 , Sox2 , Nanog ) and cell proliferation-related genes ( Pcna All of these genes were normally expressed in isolated and purified muscle satellite cells, exhibiting clear bands and good amplification specificity. Among them, muscle differentiation-related genes (… Myog , Myod1 , Desmin The positive expression of ) indicates that the isolated cells possess the potential to differentiate into muscle cells, consistent with the differentiation characteristics of muscle satellite cells; stem cell pluripotency-related genes ( Klf4 , Sox2 , Nanog The positive expression of ) indicates that the cells retain certain stem cell characteristics; proliferation-related genes ( Pcna The positive expression of ) proves that the cells have good in vitro proliferation ability.

[0034] The above results indicate that the present invention successfully isolated and cultured muscle satellite cells of Shaanbei white cashmere goat with good in vitro proliferation capacity, stem cell characteristics and muscle differentiation potential. The cell state and biological characteristics meet the requirements of subsequent experiments and can be used for further protein level identification research.

[0035] 3. Identification of immunofluorescent proteins To further identify the muscle satellite cells of Shaanbei white cashmere goats isolated and purified using the above methods, this study employed immunofluorescence staining to qualitatively detect classic specific markers and myogenesis-related markers of muscle satellite cells. Purified passage 5 muscle satellite cells were used, and staining was performed at a concentration of 3 × 10⁻⁶ cells / mL. 4Cells were seeded at a density of 1 cell / well in 24-well cell culture plates. When the cell confluence reached 80%, immunofluorescence identification was performed. First, cells were washed twice with sterile PBS, then fixed with 4% (w / w) tissue cell fixative at room temperature for 15 min, followed by permeabilization with 0.1% Triton X-100 solution for 10 min to fully expose intracellular antigen sites. Then, cells were blocked with 5% BSA blocking solution at room temperature for 1 h to effectively block non-specific antigen-antibody binding sites. After blocking, primary antibodies against the muscle satellite cell-specific marker Pax7, the key muscle development protein MyoD1, and MYH3 were added to the cell samples, and incubated overnight at 4°C. The next day, the primary antibodies were discarded, and the samples were thoroughly washed. The corresponding fluorescent secondary antibodies were added, and the samples were incubated at room temperature in the dark for 1 h. Finally, the samples were mounted with a DAPI-containing anti-fluorescence attenuation mounting medium, and the cell fluorescence expression was observed and photographed under an inverted fluorescence microscope. Results are shown below. Figure 3 .

[0036] Figure 3 Immunofluorescence staining results of Pax7, a stem marker of muscle satellite cells, showed that Pax7 protein was positively expressed in the cells (red fluorescence) and clearly localized with the nuclear dye DAPI (blue fluorescence). A large number of red / blue colocalization signals were visible in the Merge plot, indicating that a high proportion of the cell population expressed the stem marker Pax7 of muscle satellite cells.

[0037] Myod1, an early marker of myogenesis and a key regulator of early myogenesis, showed strong positive immunofluorescence (red fluorescence) and a high proportion of positive cells. It also showed significant co-localization with DAPI, indicating that the cells were in an early activated state of differentiation into myocytes, consistent with the characteristics of activated satellite cells.

[0038] Myh3, a marker of late muscle differentiation and myosin heavy chain 3, a marker of myotube differentiation, was also positively expressed (red fluorescence). Clear red / blue colocalization signals were visible in the Merge plot, indicating that the cells have the potential to differentiate into mature myocytes, further verifying the cells' myogenic differentiation capacity.

[0039] The above results indicate that the purified primary cells of Shaanbei white cashmere goats highly express Pax7, a stem cell marker for muscle satellite cells, and positively express Myod1 and Myh3, key regulatory proteins for muscle development. This further confirms at the protein level that the isolated and purified cells are genuine muscle satellite cells from Shaanbei white cashmere goats. This cell model exhibits high purity and stable biological characteristics, making it suitable for subsequent cell experiments and mechanistic studies.

[0040] This invention is not limited to the specific embodiments described above. Any modifications made by those skilled in the art based on the above concept without creative effort are within the scope of protection of this invention.

Claims

1. A method for isolating and purifying high-purity muscle satellite cells from Shaanbei white cashmere goats, characterized in that, Includes the following steps: S1. Sample aseptic pretreatment: Under aseptic conditions, the longissimus dorsi muscle tissue of fetuses of Shaanbei white cashmere goats aged 110-130 days was selected. The tissue surface was disinfected with ethanol and then thoroughly rinsed with sterile PBS containing double antibodies. S2. Tissue block inverted adherence culture: The pretreated muscle tissue is cut into uniform mince and spread evenly at the bottom of the culture dish. The culture dish is inverted and incubated for 3-5 hours. After the culture dish is upright, complete culture medium is added for constant temperature culture. S3. Primary cell passage culture: When the rate of cell migration from the edge of the tissue block reaches 35% to 45%, remove the residual tissue debris and change the complete culture medium regularly; when the cell confluence reaches 75% to 85%, wash with PBS, digest with trypsin, stop digestion with serum-containing medium, centrifuge, and then resuspend for passage. S4. Differential Adhesion Purification: When the confluence of primary cells reaches 65%–75%, the cells are digested with trypsin at room temperature, digestion is terminated with complete culture medium, and the cells are collected by centrifugation. After resuspending in fresh culture medium, the cells are passaged at a 1:1 ratio and allowed to adhere at room temperature for 25–35 minutes. The difference in adhesion rate between fibroblasts and muscle satellite cells is used to remove contaminating cells. After differential adhesion purification culture, muscle satellite cells of Shaanbei white cashmere goat are obtained.

2. The separation and purification method according to claim 1, characterized in that, In step S4, the specific steps of the graded differential adhesion purification are as follows: each generation before the 4th passage is purified by single adhesion, and in the 5th passage, the suspended cells are collected and transferred to a new sterile culture dish. The collection and purification are repeated 3 times at 25-35 min intervals.

3. The separation and purification method according to claim 1, characterized in that, In step S1, the bispecific antibody is a penicillin-streptomycin mixture with a concentration of 1%.

4. The separation and purification method according to claim 1, characterized in that, In step S2, the complete culture medium is prepared by mixing DMEM / F12 medium, fetal bovine serum and penicillin-streptomycin solution.

5. The separation and purification method according to claim 1, characterized in that, In step S2, the temperature for the isothermal incubation is 37°C.

6. The separation and purification method according to claim 1, characterized in that, In step S3, the complete culture medium is replaced every 1 to 3 days.

7. The separation and purification method according to claim 1, characterized in that, The procedure also includes RT-PCR molecular identification of the obtained muscle satellite cells: when the cell confluence reaches 55%–65%, total RNA is extracted from the cells using the TRIzol method, and the target genes are amplified by RT-PCR and detected by electrophoresis; the target genes include muscle differentiation genes, stem cell pluripotency genes, and proliferation-related genes; among which, muscle differentiation genes are… Myog , Myod1 , Desmin Stem cell pluripotency gene is Klf4 , Sox2 , Nanog The proliferation-related genes are Pcna .

8. The separation and purification method according to claim 7, characterized in that, The reaction conditions for the RT-PCR amplification were as follows: pre-denaturation at 95℃ for 5 min; 35 amplification cycles were set, with single cycle parameters as follows: denaturation at 94℃ for 30 s, annealing at 55~60℃ for 30 s, extension at 72℃ for 30 s; after the cycle, final extension at 72℃ for 10 min, and storage at 4℃.

9. The separation and purification method according to claim 1, characterized in that, The procedure also includes an immunofluorescence identification step for the obtained muscle satellite cells; the immunofluorescence identification operation includes: seeding and culturing the purified muscle satellite cells, followed by fixation, permeabilization, blocking, and antibody incubation to complete cell identification; wherein the cell seeding and culture conditions are: 3 × 10⁻⁶ cells / mL. 4 Cells were seeded at a density of 1 cell per well in 24-well plates and cultured until the cell confluence reached 75%–85%. The steps for cell fixation, permeabilization and blocking were as follows: cells were washed with sterile PBS, fixed in 4% tissue cell fixative at room temperature, permeabilized in 0.1% Triton X-100 solution, and blocked in 5% BSA blocking solution at room temperature.

10. The separation and purification method according to claim 9, characterized in that, In the immunofluorescence identification, the antibody incubation conditions were as follows: Pax7, MyoD1, and MYH3 primary antibodies were incubated overnight at 4°C, and the fluorescent secondary antibody was incubated at room temperature in the dark for 60 minutes.

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