Sample preparation

(1) Sampling: When collecting tissue samples from animals or humans, it is important to control the volume of the tissue block. This volume should be kept within an appropriate range. Doing so ensures that the fixative can penetrate the tissue evenly and completely.

(2)PBS/saline rinse: Remove blood or other body fluids in the tissue sample that may affect the fixation effect.

(3)Fixation: Fix with 10% formalin, methanol, ethanol, or a mixture thereof at room temperature for 18-24 hours. Ensure that the tissue sample is completely immersed in the fixative to avoid unfixed parts.

(4)Post-fixation treatment: rinse off the fixative with running water.

Commonly used embedding media include paraffin, resin, etc. Take paraffin as an example.

(1)Dehydration: Alcohol gradient dehydration, soak in 75% ethanol, 85% ethanol, 95% ethanol, anhydrous ethanol (I), and anhydrous ethanol (II) for 30-60 minutes respectively. The sample will become opaque at this time.

(2)Transparency: Soak in a transparent agent (such as xylene) twice, each time for 30 minutes to remove alcohol and other solvents. Be careful to avoid excessive transparency, which may cause the sample to become hard and brittle.

(3)Wax immersion: Immerse the transparentized tissue sample in molten paraffin and place it in a constant temperature box for immersion, so that the paraffin can fully and evenly penetrate the tissue.

(4)Embedding: Place the wax-immersed tissue sample into a mold and pour in molten paraffin. Wait for the paraffin to cool and solidify, and avoid excessive cooling that may cause the paraffin to break or the tissue to deform.

Sectioning

(1)Sectioning: Cut the embedded tissue sample into 4-5 μm thick slices using a microtome.

(2)Slice baking: Gently separate the slices from the slicer with a fine brush, then place them in 40℃ warm water to fully flatten them. Finally, bake them at 60℃ for 2 hours to enhance the adhesion of the slices.

Dewaxing and rehydration

(1)Dewaxing: Dewaxing was performed using xylene I, II, and III in a gradient manner, with each chamber soaking for 5 minutes. Thorough removal of paraffin is crucial for subsequent experiments, as any residue will interfere with antigen exposure and staining.

(2)Rehydration: Dehydrate using gradient ethanol (100% ethanol I, 100% ethanol II, 95% ethanol, 85% ethanol, 70% ethanol) for 5 minutes each; finally, immerse in purified water for 5 minutes to complete the hydration process. Note that drying of the slides should be avoided, as drying of the slides will result in high background due to nonspecific antibody binding.

Antigen retrieval

Antigen retrieval techniques are employed to expose blocked epitopes, thereby enhancing antibody binding efficiency. Heat-induced antigen retrieval is the most commonly used method.The following retrieval buffers are recommended: citrate buffer (pH 6.0), EDTA buffer (pH 8.0), or Tris-EDTA buffer (pH 9.0). If the citrate buffer proves ineffective, EDTA and Tris-EDTA buffers can be used. However, it is important to note that these buffers may lead to nonspecific staining in samples with high expression levels.

To achieve optimal staining results, it is advisable to try several retrieval methods.

(1) Microwave heat repair: Add antigen repair solution, put in the slices, place in the microwave oven on medium heat for 8 minutes, turn off the heat for 7 minutes, turn to low heat for 8 minutes; remove from the staining box and cool to room temperature.

(2)Water bath heat repair: Add antigen repair solution, put in the slices, place in a water bath at 95°C for 25-30 minutes (pay attention to control the temperature to avoid boiling to prevent tissue shedding). Cool the sample and water together, do not cool suddenly to avoid rapid cooling that will cause changes in the antigen epitope conformation.

(3)High pressure heat repair: Add antigen repair solution to the staining box, put in the slices, place in a pressure cooker and heat to full pressure, then continue heating for 5 minutes, turn off the power, remove from the staining box after 10 minutes, and cool to room temperature.

(4)Enzymatic repair: Commonly used digestive enzymes include trypsin, pepsin, proteinase K, etc. Place the slices on a slide containing enzymatic solution, and then perform enzymatic hydrolysis under appropriate temperature and humidity conditions (usually 37°C, 20 minutes). After enzymatic hydrolysis, the slices also need to be cooled to room temperature before subsequent processing. After repair, wash three times with PBS on a shaker for 5 min each time.

Blocking

(1)Primary Antibody Dilution: Select an appropriate primary antibody and dilute it with a diluent (PBS, TBS, etc.) to an appropriate ratio.

(2)Primary Antibody Incubation: Use a pipette to evenly cover the sample area with the diluted primary antibody, place the sample in a humidity-balanced incubation box, and incubate at 37°C for 90 minutes to promote antigen-antibody specific binding.

(3)Washing: Wash three times with PBS on a shaker for 5 minutes each. For samples that are prone to detachment, use a mild elution buffer pre-warmed at 37°C and gently wash for 5-20 minutes to maintain tissue integrity while effectively removing non-specific binding.

Secondary antibody incubation

(1)Secondary Antibody Dilution: Select an appropriate secondary antibody and dilute it to the appropriate ratio using diluent (PBS, TBS, etc.).

(2)Secondary Antibody Incubation: Incubate the secondary antibody at room temperature for 1 h, protecting the slides from light and allowing them to dry.

(3)Washing: Wash the slides three times with PBS on a shaker for 5 minutes each.

TSA dye incubation

(1)Dilute TSA: Dilute TSA dye 1:50-1:200 with 1× TSA buffer to prepare a working solution. Store the staining solution at room temperature in the dark for up to 24 hours.

(2)Add 100 μL of the working staining solution to the slide, submerge the sample, and incubate on a shaker at room temperature for 5-10 minutes, taking care to avoid drying.

(3)Wash: Wash three times with PBS on a shaker for 5 minutes each.

Antibody shedding

The steps are the same as those for the antigen retrieval step above, but the purpose is to wash away the incubated antibodies.

For frozen sections, cell slides, and bone tissue (which tend to detach easily), it is recommended to add an appropriate amount of mIHC antibody elution buffer, preheated to 37°C until completely dissolved, to evenly cover the sample. Incubate at 37°C for 5-20 minutes, discard the elution buffer, and then add an appropriate amount of antibody elution buffer to evenly cover the sample. Incubate at 37°C for 5-20 minutes.

Washing

Wash once with ddH2O and soak the slides in PBS for 2 min.

Repeat

To seal the slide or continue staining, start from the "Blocking" step. Repeat the steps including blocking endogenous peroxidase - blocking - primary antibody incubation - secondary antibody incubation - TSA fluorescent dye staining - antibody elution.

Nuclear staining and sealing

Add DAPI working solution to the sample and incubate at room temperature for 5 minutes. Wash once with PBS and mount with anti-fluorescence quenching mounting medium. For long-term storage, it is recommended to seal the edges of the coverslip with clear nail polish.

The sections were observed and images were collected under a fluorescence microscope/confocal microscope/multi-channel fluorescence scanner/multispectral imaging system.

6.What should I do if dewaxing is incomplete and the tissue shows metachromatic staining?Adjust the dewaxing time based on the room temperature. The key is to ensure a thorough and clean finish. In the summer, when the room temperature is high, dewaxing time is usually shorter, 3-5 minutes is sufficient. In the winter, when the room temperature is lower, dewaxing time may need to be extended, to 10-20 minutes or even longer.

7.How can I reduce nonspecific staining?

Shorten the primary/secondary antibody incubation time and dilute the antibody ratio. Polyclonal primary antibodies are prone to background, so monoclonal antibodies can be used. Endogenous catalase levels are high in tissues such as the liver and kidney, so the blocking time should be extended to reduce background. To prevent nonspecific components from binding to the antibody, extend the blocking time of the animal immune serum used to generate the secondary antibody. Increase the number and duration of PBS washes as appropriate. Prevent the slides from drying out during the experiment.

Do we need to do hotfixes every time?

The process before the first antibody staining is called antigen retrieval. This is done to expose antigenic determinants blocked by paraffin or fixative, allowing the antibody to recognize the antigen. Antigen retrieval before subsequent rounds of antibody staining aims to remove antibodies from the previous round of staining as well as any unbound dye. For fresh, frozen sections, antigen retrieval is not necessary, but endogenous peroxidase blocking is still necessary. Frozen sections of fixed tissue still require antigen retrieval, but heating frozen sections can cause detachment, so we generally do not recommend switching fixed samples to frozen sections. Alternatively, frozen sections can be treated with antigen elution buffer.