Preparation and detection method of multi-label immunohistochemical bone marrow microenvironment sample

Abstract

The invention provides a preparation and detection method of a multi-label immunohistochemical bone marrow microenvironment sample, and overcomes the traditional technical defects caused by factors such as interference of non-specific signals and conflict of antigen species sources. According to the preparation and detection method of the multi-label immunohistochemical bone marrow microenvironment sample, a complete and high-quality bone tissue paraffin section is obtained through optimization of the preparation method in the preparation process of the bone tissue paraffin section; and a complete thighbone / tibia slice panoramic image can still be presented after a multicolor labeling immunohistochemical method is optimized. According to the invention, the purpose of real-time detection and analysis of mutual expressions of hematopoietic stem cells, immune cells, mesenchymal stem cells, microvessels, various related cell factors and chemotactic factors and the like in bone tissue and bone marrow microenvironments can be realized; and the method has important practical application value for the steady state maintenance of the microenvironment of bone tissues and bone marrow and the research on the occurrence and progress of diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a method for preparing and detecting a multi-labeled immunohistochemical bone marrow microenvironment sample. Background Art

[0002] Flow cytometry, sequencing, and protein microarrays are commonly used to analyze the bone marrow microenvironment. These methods can capture an increasing number of genotypes or cell-type components, but they cannot reveal the in situ distribution characteristics of specific phenotypes within the tissue. The spatial localization of cells or key molecules and their relationship with tissue structure are crucial for understanding the roles and effects of each component within the tissue microenvironment.

[0003] Immunohistochemistry, also known as immunocytochemistry, involves the use of specific antibodies labeled with chromogens to qualitatively, locally, and quantitatively determine the specific antigens in situ in tissue cells through antigen-antibody reactions and histochemical color development. It cleverly combines the specificity of immune responses with the visibility of histochemistry, leveraging the imaging and magnification of fluorescence or electron microscopy to detect various antigens, including proteins, peptides, enzymes, hormones, pathogens, and receptors, at the cellular and subcellular levels. While immunohistochemical staining can address multiple requirements, including protein quantification, spatial localization, and tissue structural relationships, the application of traditional immunohistochemical methods in the bone marrow microenvironment is significantly limited by interference from nonspecific signals and conflicting antigen species.

[0004] The novel multiplex immunohistochemistry (mIHC) method, derived from tyramide signal amplification technology, can overcome the limitations of antigen species and enable the simultaneous detection of up to seven molecular targets in the same tissue sample. This is crucial for the precise study of the immune microenvironment. Currently, this method has been widely used in the analysis of the tumor immune microenvironment of solid tumors and has been shown to have higher diagnostic accuracy in predicting response to anti-PD-1 / PD-L1 immunotherapy. However, the application of this method in the study of hematological tumors and the bone marrow microenvironment has rarely been reported, which is one of the important issues that need to be addressed in this field. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the present invention aims to establish a complete set of preparation and detection methods for multi-labeled immunohistochemical samples suitable for the bone marrow microenvironment based on the characteristics of the bone marrow microenvironment and tyramide signal amplification technology.

[0006] In a first aspect of the present invention, a method for preparing a bone marrow microenvironment sample for multi-labeled immunohistochemistry is provided, characterized in that the method includes preparing bone tissue paraffin sections and preparing multi-color labeled immunohistochemistry samples, specifically comprising the following steps:

[0007] 1. Preparation of paraffin sections of bone tissue:

[0008] (1) Take bone tissue and place it in buffer solution for later use;

[0009] (2) Fixation: Place the obtained bone tissue in a fixative and fix it at room temperature to allow the fixative to penetrate the bone tissue; then change the temperature to 35-40°C to fix the bone marrow tissue quickly;

[0010] (3) Decalcification: The fixed bone tissue is washed with water (ddH2O) and then placed in EDTA decalcification solution. Decalcification is carried out at room temperature, and the decalcification solution is changed in the morning and evening until the bone becomes soft and decalcification is completed.

[0011] (4) Dehydration, transparency and wax immersion: The decalcified bone tissue is dehydrated and transparentized by ethanol and xylene at room temperature; the tissue is then paraffinized to make wax blocks for later use;

[0012] (5) Embedding: Pour liquid paraffin into a container, then place the wax-soaked tissue block flat on the bottom. After the paraffin solidifies, remove the embedding frame. After it completely cools and hardens, trim the wax block. Keep the paraffin around the tissue to a moderate level for slicing.

[0013] (6) Sectioning: Prepare paraffin sections using a microtome, spread the sections in warm water, and use a glass slide with an adhesive coating to pick up the sections for later use;

[0014] 2. Preparation of multi-color immunohistochemistry samples:

[0015] (7) The prepared paraffin sections are placed in a constant temperature oven for baking. Freshly prepared paraffin sections should be stained within one month to ensure antigen activity.

[0016] (8) Dewaxing and hydration: The baked paraffin sections were dewaxed with xylene, then hydrated with ethanol, washed with water (ddH2O), and fixed again in a fixative.

[0017] (9) Antigen retrieval: After the fixed sample is washed with water (ddH2O), the sample is placed in a buffer solution and retrieval is performed in a water bath;

[0018] (10) Blocking peroxidase: Wash the sample with buffer after repair treatment and add endogenous peroxidase blocker to block at room temperature;

[0019] (11) Blocking: Wash the blocked sample with buffer solution and add immunostaining blocking solution to block at room temperature;

[0020] (12) Primary antibody incubation: Use a monoclonal antibody specifically for immunohistochemistry as the primary antibody marker; incubate the primary antibody at room temperature;

[0021] (13) Secondary antibody incubation: Wash the sample with buffer after primary antibody incubation, add secondary antibody, and incubate at room temperature in the dark;

[0022] (14) TSA signal amplification: Wash the sample after incubation with secondary antibody with buffer solution, add TSA signal amplification solution, incubate at room temperature in the dark, and wash with buffer solution after incubation;

[0023] (15) The first round of staining is completed above. The number of staining times is determined according to the number of labeled primary antibodies. Steps (9) to (14) are repeated to complete the staining. Then, DNA fluorescent dye is added dropwise, and the buffer solution is washed. Anti-fluorescence quenching mounting medium is added dropwise, and the slide is sealed with a coverslip for later use.

[0024] Furthermore, the bone tissue is mouse bone tissue.

[0025] Furthermore, in step (1), the bone tissue is femur and tibia, and the buffer is PBS buffer.

[0026] Furthermore, in step (2), the fixative is 10% neutral formalin, the ratio of the fixative to the tissue is 1:40-60 (preferably 1:50), the room temperature fixation time is 20-28 hours (preferably 24 hours), the temperature is changed to 37°C, and the fixation time is 68-76 hours (preferably 72 hours).

[0027] Furthermore, in step (3), the EDTA decalcification solution is a commercial neutral EDTA decalcification solution (Solarbol), the room temperature decalcification time is 30-45 days (preferably 35-40 days), and the amount of replacement decalcification solution is 3 ml / root bone tissue.

[0028] 1. Furthermore, in step (4), the ethanol and xylene treatments are specifically 50%, 60%, 75%, 85%, and 95% ethanol for 1 hour each, 100% ethanol I and II for 1 hour each, xylene I for 1 hour, and xylene II for 1 hour; and the paraffin treatment is specifically paraffin I for 30 minutes, paraffin II for 30 minutes, paraffin III for 1 hour, and paraffin IIII for 1 hour.

[0029] Furthermore, in step (6), the thickness of the paraffin section is 2-3 μm, and the temperature of the warm water is 40-50° C. (preferably 45° C.).

[0030] Furthermore, in step (7), the temperature in the thermostat is 50-70° C. (preferably 60° C.), and the baking time is 50-70 min (preferably 60 min).

[0031] Furthermore, in the step (8), the dewaxing with xylene is specifically performed by treating with xylene I for 15 minutes and treating with xylene II for 5 minutes; the hydration with ethanol is specifically performed by hydrating with anhydrous ethanol for 3 minutes, anhydrous ethanol for 3 minutes, 95% ethanol for 3 minutes, 95% ethanol for 3 minutes, 80% ethanol for 3 minutes, and 70% ethanol for 3 minutes in sequence; the fixing liquid is 10% neutral formalin, and the fixing time is 10-25 minutes (preferably 15-20 minutes).

[0032] Furthermore, in step (9), the buffer is sodium citrate buffer (pH=6) or EDTA buffer (pH=9), the water bath temperature is 90-99°C (preferably 95-99°C), and the repair time is 10-25 min (preferably 15-20 min).

[0033] Furthermore, in step (10), the buffer solution is PBST, and the specific washing times and time are 3 washes, each time for 5 minutes; the room temperature blocking time is 5-20 minutes (preferably 10-15 minutes).

[0034] Furthermore, in step (11), the buffer solution is PBS, and the specific washing times and time are 3 washes, each time for 5 minutes; the room temperature blocking time is 15-25 minutes (preferably 20 minutes).

[0035] Furthermore, in step (12), the selected immunohistochemistry-specific monoclonal antibodies include rabbit Anti-c-Kit antibody (Abcam), rabbit Anti-CD150 antibody (Abcam), rabbit Anti-Laminin antibody (Abcam), rabbit Anti-Sca-1 antibody (Abcam) and rabbit Anti-LepR antibody (Invitrogen), only one antibody is labeled in a single round and labeled in the order of the above antibodies; the primary antibody incubation is at a concentration of 1:200, and the incubation is at room temperature for 1 hour or at 4°C overnight.

[0036] Furthermore, in step (13), the buffer solution is PBST, the specific washing times and time are 3 washes, each time for 5 minutes; the secondary antibody is goat anti-rabbit IgG HRP secondary antibody; and the dark-proof room temperature incubation time is 10 to 20 minutes.

[0037] Furthermore, in the step (14), the buffer solution is PBST, and the specific number and time of washing are 3 washes, each time for 5 minutes; when adding TSA signal amplification liquid, only one TSA signal amplification liquid is added in a single round, and the order is TSA690, TSA650, TSA570, TSA520, and TSA480; the light-proof room temperature incubation time is 5 to 15 minutes (preferably 10 minutes), and the buffer solution for the final wash is PBST, and the specific number and time of washing are 2 washes, each time for 5 minutes.

[0038] Furthermore, in step (15), the DNA fluorescent dye is DAPI, and the buffer is PBS.

[0039] Furthermore, the six biomarkers marked in the method are HSCs (c-Kit + 、Sca-1 + 、CD150 + ), bone marrow microenvironment stromal cells (mesenchymal stem cells: LepR + ; Vascular endothelial cells: Laminin + ) and nuclei DAPI.

[0040] In a second aspect of the present invention, a multi-marker immunohistochemical bone marrow microenvironment sample is provided, characterized in that it is prepared by the method for preparing a multi-marker immunohistochemical bone marrow microenvironment sample according to the first aspect of the present invention.

[0041] In the third aspect of the present invention, a method for detecting a multi-labeled immunohistochemical bone marrow microenvironment sample is provided, characterized in that a microscopic imaging system is used to image and detect the multi-labeled immunohistochemical bone marrow microenvironment sample described in the second aspect of the present invention.

[0042] Furthermore, the microscopic imaging system is a combination of excitation light and filters of the microscopic imaging system Ex360 / DAPI, EX450 / EM480, EX490 / EM520, EX550 / EM570, EX630 / EM650, EX680 / EM690.

[0043] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one.

[0044] Compared with the prior art, the present invention has the following advantages and improvements:

[0045] The present invention establishes a complete set of preparation and detection methods for multi-labeled immunohistochemical samples suitable for bone marrow microenvironment, overcoming the traditional technical defects caused by factors such as interference from non-specific signals and conflicts in antigen species sources.

[0046] First, the preparation and detection method of the multi-labeled immunohistochemical bone marrow microenvironment sample of the present invention obtains complete and high-quality bone tissue paraffin sections by optimizing the preparation method during the preparation of bone tissue paraffin sections. Specifically, conventional tissue fixation methods are insufficient for bone tissue, and most of the bone marrow in the diaphysis is lost due to insufficient fixation; even if the fixation time is extended to 72 hours, the situation does not improve; and the bone tissue fixation method of the present invention is significantly superior to conventional tissue fixation methods. At the same time, compared with the HE staining of hydrochloric acid and formic acid decalcified paraffin sections commonly used in clinical testing, EDTA decalcification better preserves nucleic acid and protein structures, while the nucleic acid and protein structures are destroyed after hydrochloric acid and formic acid decalcification, resulting in no cell nucleus staining in HE staining and low peaks of immunohistochemical protein markers. In the dehydration step, the present invention reduces the ethanol concentration after the sample is fully fixed, starts dehydration from a milder low-concentration ethanol, and uses room temperature dehydration to reduce the probability of abnormal bone marrow tissue sections.

[0047] Secondly, the present invention optimizes the multicolor immunohistochemistry method to still produce complete panoramic images of femoral / tibia slices. Specifically, during the preparation of multicolor immunohistochemistry samples, the bone marrow microenvironment is fully visualized by optimizing the dewaxing, hydration, and antigen retrieval steps. Compared to microwave oven or pressure cooker heat retrieval, the present invention's antigen retrieval method is gentler in a water bath and effectively prevents bone tissue from detaching. Compared to pepsin retrieval, the water bath method is more applicable to a wider range of antigens and does not damage antigen epitopes or tissue structure.

[0048] Finally, the present invention can realize the application of multi-marker immunohistochemistry to prepare samples in bone tissue and bone marrow microenvironment, and achieves the purpose of simultaneous real-time detection and analysis of the mutual expression of hematopoietic stem cells, immune cells, mesenchymal stem cells, microvessels (endothelial cells) and various related cytokines and chemokines in bone tissue and bone marrow microenvironment. It has significant practical application value for the study of bone tissue and bone marrow microenvironment homeostasis maintenance, disease occurrence and progression. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0050] Figure 1 HE staining results of paraffin sections of mouse femur and tibia. The sections were fixed in 10% neutral formalin for 24 hours at room temperature and 72 hours at 37°C.

[0051] Figure 2 : HE staining results of paraffin sections of mouse femurs. The sections were fixed in 10% neutral formalin at room temperature for 24 hours.

[0052] Figure 3 : HE staining results of paraffin sections of mouse femurs. The sections were fixed in 10% neutral formalin at room temperature for 72 hours.

[0053] Figure 4 HE staining of mouse bone marrow paraffin sections. Left: EDTA decalcification for 30 days; right: hydrochloric acid-formic acid decalcification for 5 days.

[0054] Figure 5 HE staining of mouse bone marrow paraffin sections. Left: Dehydration starting with 75% ethanol; right: Dehydration starting with 50% ethanol.

[0055] Figure 6 :Tissue staining results after water bath antigen retrieval (red box marked image see Figure 7 ).

[0056] Figure 7 : Figure 6 The images marked with red frames, A is the image merged with 6 colors, B is the image merged with 4 colors, and C is the image merged with two colors. DETAILED DESCRIPTION

[0057] The present invention provides a method for preparing and detecting a multi-labeled immunohistochemical bone marrow microenvironment sample. The present invention is described in detail below in conjunction with the examples to facilitate further understanding of the present invention by those skilled in the art. However, the examples described below are only part of the examples of the present invention and should not be regarded as any form of limitation to the present invention. It should be pointed out that adjustments and improvements made by those of ordinary skill in the art based on the concept of the present invention should be regarded as the scope of protection of the present invention. Specific technical operation steps and operators are not indicated in the examples, and are carried out in accordance with the general technical conditions described in the literature of this field or the relevant product instructions.

[0058] Example

[0059] This embodiment proposes a method for preparing and imaging bone marrow microenvironment samples using multi-labeled immunohistochemistry based on tyramide signal amplification technology, including (1) bone marrow sample preparation; (2) fluorescent labeling sample preparation; and (3) microscopic imaging.

[0060] Sample preparation is key to accurately obtaining information about the bone marrow microenvironment, especially the steps of preparing intact mouse bone marrow paraffin sections and fluorescently labeling the sections.

[0061] 1. Preparation of paraffin sections of mouse bone tissue:

[0062] (1) Wild-type C57 male mice (8–10 weeks old) were purchased from the Laboratory Animal Center of the Hematology Hospital of the Chinese Academy of Medical Sciences (Institute of Hematology, Chinese Academy of Medical Sciences). Mice were euthanized using carbon dioxide, and bilateral femurs and tibias were removed and placed in PBS buffer for later use.

[0063] (2) Fixation: Place the fresh sample in 50 ml of 10% neutral formalin (fixative to tissue ratio is 1:50) and fix it at room temperature for 24 hours to allow the fixative to penetrate the bone tissue; then change to 37°C and fix it for 72 hours to allow the bone marrow tissue to be quickly fixed in a short time ( Figure 1 );

[0064] The general recommendation for tissue fixation is 10% neutral formalin or 4% paraformaldehyde at room temperature for 12-24 hours, but this is not sufficient for mouse bone tissue ( Figure 2 ), most of the bone marrow in the diaphysis was lost due to insufficient fixation. Extending the fixation time to 72 hours did not improve the situation ( Figure 3 ). It can be seen that the bone tissue fixation method of the present invention is significantly superior to the conventional tissue fixation method.

[0065] (3) Decalcification: The fixed specimens were washed with ddH2O and then placed in a commercial neutral EDTA decalcification solution (Solarbo) for 35–40 days at room temperature. The decalcification solution was changed in the morning and evening at a rate of 3 ml per root until the bone softened and decalcification was complete.

[0066] Comparison of HE staining of paraffin sections decalcified with EDTA and hydrochloric acid and formic acid commonly used in clinical testing ( Figure 4 ), EDTA better preserves nucleic acid and protein structures, while decalcification with hydrochloric acid and formic acid destroys nucleic acid and protein structures, resulting in no nuclear staining in HE staining and low peaks of immunohistochemical protein labeling.

[0067] (4) Dehydration, transparency, and wax immersion: The decalcified samples were treated with 50%, 60%, 75%, 85%, and 95% ethanol for 1 hour each, 100% ethanol I and II for 1 hour each, xylene I for 1 hour, and xylene II for 1 hour at room temperature for dehydration and transparency. The tissues were then treated with paraffin I for 30 minutes, paraffin II for 30 minutes, paraffin III for 1 hour, and paraffin IIII for 1 hour to prepare wax blocks for later use.

[0068] Bone and bone marrow have different densities and bone tissue contains a lot of water. Improper dehydration can easily cause the bone marrow to shrink and separate from the bone; or the tissue may break into pieces. Therefore, choosing the right dehydration concentration gradient is very important. Clinical pathology usually starts with a higher concentration of ethanol for soft tissue dehydration and heats the dehydration process, which can easily cause the bone marrow to shrink. Therefore, after the sample is fully fixed, the ethanol concentration should be reduced, and dehydration should be started from a milder low concentration of ethanol. Dehydration at room temperature can reduce the probability of abnormal bone marrow tissue sections ( Figure 5 ).

[0069] (5) Embedding: Pour liquid paraffin into the mold box, then place the wax-soaked tissue block flat on the bottom, with the cut surface facing downward. After the paraffin solidifies, remove the embedding frame. After it is completely cooled and hardened, trim the wax block. Keep the paraffin around the tissue to a moderate level for slicing.

[0070] (6) Sectioning: Use a microtome to prepare paraffin sections with a thickness of 2-3 μm, spread the sections at 45°C, and use a glass slide with an adhesive coating to pick up the sections for later use.

[0071] 2. Preparation and detection of multi-color immunohistochemistry samples:

[0072] (1) Place the paraffin sections in a 60°C constant temperature oven and bake for at least 60 minutes. Freshly prepared paraffin sections should be stained within one month to ensure antigen activity.

[0073] (2) Dewaxing and hydration: Samples were treated with xylene I for 15 min and xylene II for 5 min for dewaxing. They were then hydrated with anhydrous ethanol for 3 min, anhydrous ethanol for 3 min, 95% ethanol for 3 min, 95% ethanol for 3 min, 80% ethanol for 3 min, and 70% ethanol for 3 min. After washing with ddH2O, the samples were fixed again in 10% neutral formalin for 15–20 min.

[0074] (3) Antigen repair: After washing with ddH2O, place the sample in sodium citrate buffer (pH=6) or EDTA buffer (pH=9) and repair it in a water bath at 95℃~99℃ for 15-20 minutes. Compared with microwave oven or pressure cooker heat repair method, water bath method is gentler and can effectively prevent bone tissue from peeling off ( Figure 6 and Figure 7 ); Compared with the pepsin repair method, the water bath method is applicable to a wider range of antigens and will not destroy the antigen epitope or tissue structure.

[0075] (4) Blocking peroxidase: Wash with PBST for 5 minutes three times, and add endogenous peroxidase blocker to block at room temperature for 10 to 15 minutes.

[0076] (5) Blocking: Wash with PBS for 5 minutes*3 times, add immunostaining blocking solution (goat serum) and block at room temperature for 20 minutes.

[0077] (6) Primary antibody incubation: Monoclonal antibodies specifically designed for immunohistochemistry were used as primary antibody markers, including rabbit anti-c-Kit antibody (Abcam), rabbit anti-CD150 antibody (Abcam), rabbit anti-Laminin antibody (Abcam), rabbit anti-Sca-1 antibody (Abcam), and rabbit anti-LepR antibody (Invitrogen). Only one antibody was labeled per round, and the labels were labeled in the order listed above. Primary antibody incubation was performed at a concentration of 1:200 for 1 hour at room temperature or overnight at 4°C.

[0078] (7) Secondary antibody incubation: Wash with PBST for 5 min*3 times, add goat anti-rabbit IgG HRP secondary antibody, and incubate at room temperature in the dark for 10-20 min.

[0079] (8) TSA signal amplification: Wash with PBST for 5 min*3 times, add TSA signal amplification solution (only one TSA signal amplification solution is added in a single round, the order is TSA690, TSA650, TSA570, TSA520, TSA480), incubate at room temperature in the dark for 10 min, and wash with PBST for 5 min*2 times.

[0080] The above is the first round of staining. The number of staining times is determined according to the number of labeled primary antibodies. Repeat steps (3) to (8) to complete the staining. Then add DAPI, wash with PBS, add anti-fluorescence quenching mounting medium, and cover the slide with a coverslip for later use. This method labels six biomarkers: HSCs (c-Kit + Sca-1 + CD150 + ) and bone marrow microenvironment stromal cells (mesenchymal stem cells: LepR + ; Vascular endothelial cells: Laminin + ) and nuclei DAPI.

[0081] 3. Microscopic imaging

[0082] The stained sections were imaged using the microscope imaging system Ex360 / DAPI, EX450 / EM480, EX490 / EM520, EX550 / EM570, EX630 / EM650, and EX680 / EM690 excitation light and filter combinations.

[0083] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. For those skilled in the art, any modifications and changes made to the above embodiment based on the technical essence of the present invention should fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for preparing a bone marrow microenvironment sample for multi-label immunohistochemistry, characterized in that: The preparation of paraffin sections of bone tissue and multi-color immunohistochemistry samples includes the following steps: A) Preparation of paraffin sections of bone tissue: (1) Take bone tissue and place it in buffer solution for later use; (2) Fixation: Place the obtained bone tissue in a fixative and fix it at room temperature to allow the fixative to penetrate the bone tissue; then change the temperature to 35-40°C to fix the bone marrow tissue quickly; (3) Decalcification: The fixed bone tissue is washed with water (ddH2O) and then placed in EDTA decalcification solution. Decalcification is carried out at room temperature, and the decalcification solution is changed in the morning and evening until the bone becomes soft and decalcification is completed. (4) Dehydration, transparency and wax immersion: The decalcified bone tissue is dehydrated and transparentized by ethanol and xylene at room temperature; the tissue is then paraffinized to make wax blocks for later use; (5) Embedding: Pour liquid paraffin into a container, then place the wax-soaked tissue block flat on the bottom. After the paraffin solidifies, remove the embedding frame. After it completely cools and hardens, trim the wax block. Keep the paraffin around the tissue to a moderate level for slicing. (6) Sectioning: Prepare paraffin sections using a microtome, spread the sections in warm water, and use a glass slide with an adhesive coating to pick up the sections for later use; B) Preparation of samples for multi-color immunohistochemistry: (7) The prepared paraffin sections are placed in a constant temperature oven for baking. Freshly prepared paraffin sections should be stained within one month to ensure antigen activity. (8) Dewaxing and hydration: The baked paraffin sections were dewaxed with xylene, then hydrated with ethanol, washed with water (ddH2O), and fixed again in a fixative. (9) Antigen retrieval: After the fixed sample is washed with water (ddH2O), the sample is placed in a buffer solution and retrieval is performed in a water bath; (10) Blocking peroxidase: Wash the sample with buffer after repair treatment and add endogenous peroxidase blocker to block at room temperature; (11) Blocking: Wash the blocked sample with buffer solution and add immunostaining blocking solution to block at room temperature; (12) Primary antibody incubation: Use a monoclonal antibody specifically for immunohistochemistry as the primary antibody marker; incubate the primary antibody at room temperature; (13) Secondary antibody incubation: Wash the sample with buffer after primary antibody incubation, add secondary antibody, and incubate at room temperature in the dark; (14) TSA signal amplification: Wash the sample after incubation with secondary antibody with buffer solution, add TSA signal amplification solution, incubate at room temperature in the dark, and wash with buffer solution after incubation; (15) The first round of staining is completed as above. The number of staining times is determined according to the number of labeled primary antibodies. Steps (9) to (14) are repeated to complete the staining. Then, DNA fluorescent dye is added dropwise, and the buffer solution is washed. Anti-fluorescence quenching sealing agent is added dropwise, and the slide is sealed with a coverslip for later use.

2. The method for preparing a bone marrow microenvironment sample by multi-label immunohistochemistry according to claim 1, characterized in that: In the step (2), the fixative is 10% neutral formalin, the ratio of the fixative to the tissue is 1:40-60, the room temperature fixation time is 20-28 hours, the temperature is changed to 37° C., and the fixation time is 68-76 hours.

3. The method for preparing a bone marrow microenvironment sample by multi-label immunohistochemistry according to claim 1, characterized in that: In the step (3), the time for decalcification at room temperature is 30-45 days.

4. The method for preparing a bone marrow microenvironment sample by multi-label immunohistochemistry according to claim 1, characterized in that: In the step (4), the ethanol and xylene treatments are specifically 50%, 60%, 75%, 85%, and 95% ethanol for 1 hour each, 100% ethanol I and II for 1 hour each, xylene for 1 hour, and xylene II for 1 hour; the paraffin treatments are specifically paraffin I for 30 minutes, paraffin II for 30 minutes, paraffin III for 1 hour, and paraffin IIII for 1 hour.

5. The method for preparing a bone marrow microenvironment sample for multi-label immunohistochemistry according to claim 1, characterized in that: In the step (7), the temperature in the constant temperature box is 50-70° C., and the baking time is 50-70 minutes.

6. The method for preparing a bone marrow microenvironment sample for multi-label immunohistochemistry according to claim 1, characterized in that: In the step (8), the dewaxing using xylene is specifically performed by treating with xylene I for 15 minutes and treating with xylene II for 5 minutes; the hydration using ethanol is specifically performed by hydrating with anhydrous ethanol for 3 minutes, anhydrous ethanol for 3 minutes, 95% ethanol for 3 minutes, 95% ethanol for 3 minutes, 80% ethanol for 3 minutes, and 70% ethanol for 3 minutes in sequence; the fixing liquid is 10% neutral formalin, and the fixing time is 10-25 minutes.

7. The method for preparing a bone marrow microenvironment sample for multi-label immunohistochemistry according to claim 1, characterized in that: In the step (9), the buffer is sodium citrate buffer or EDTA buffer, the water bath temperature is 90-99° C., and the repair time is 10-25 min.

8. The method for preparing a bone marrow microenvironment sample for multi-label immunohistochemistry according to claim 1, characterized in that: In the step (13), the secondary antibody is goat anti-rabbit IgG HRP secondary antibody; and the dark-proof room temperature incubation time is 10 to 20 minutes.

9. A multi-marker immunohistochemistry bone marrow microenvironment sample, characterized in that: The sample is prepared by the method for preparing a multi-labeled immunohistochemical bone marrow microenvironment sample according to any one of claims 1 to 8.

10. A method for detecting bone marrow microenvironment samples by multi-label immunohistochemistry, characterized in that: The multi-labeled immunohistochemical bone marrow microenvironment sample according to claim 9 is imaged and detected using a microscopic imaging system.

Images