Primary antibody and secondary antibody eluent, specially for mIHC experiments 

Primary antibody and secondary antibody eluent, specially for mIHC experiments is an eluent used for mIHC (multiple immunohistochemistry).

I. Sectioning
1. Frozen Sectioning
1.1 Remove the frozen sections, return them to room temperature, dry them, and fix them for 10-30 minutes. Wash them with PBS for 5 minutes, repeating three times.
1.2 Prepare the permeabilization solution. Permeabilize by adding permeabilization buffer for 20 minutes. Wash with PBS for 5 minutes, repeat three times.
1.3 Perform antigen retrieval. Place sections in EDTA antigen retrieval buffer (pH 9.0) in a 60°C water bath for 30 minutes. Allow to cool naturally, then wash with PBS for 5 minutes, repeat three times.
Note: This retrieval step may cause slides to delaminate. It is optional and should only be performed if the imaging background is high.
1.4 Block endogenous peroxidases. Incubate sections in 3% hydrogen peroxide solution at room temperature in the dark for 25 minutes. Wash slides three times in PBS (pH 7.4) on a destaining shaker, shaking for 5 minutes each.
1.5 BSA Blocking: After lightly shaking the sections, use a histochemical pen to draw a circle around the tissue (to prevent antibody loss). Add 3% BSA (or other blocking solution) to the circle, evenly covering the tissue. Block at room temperature for 30 minutes.

2. Antibody Incubation
2.1 Primary Antibody Incubation: Gently shake off the blocking solution. Add the primary antibody diluted in antibody diluent to the sections. Incubate the sections flat in a light-proof, humidified chamber at 4°C overnight or at 37°C for 1-2 hours. Be careful not to allow the sections to dry out.
2.2 Secondary Antibody Incubation: Wash the slides three times with PBS (pH 7.4) on a destaining shaker, 5 minutes each. After lightly shaking the sections, add the secondary antibody to the tissue, incubate at room temperature for 50 minutes, and then wash three times with PBS, 5 minutes each.

3. Staining
Mix TSA dye and TSA buffer at a ratio of 1:50-1:200. Add the prepared TSA fluorescent dye reaction solution evenly to the sections, covering the tissue. Incubate at room temperature for 1-15 minutes (optimal time is 5-10 minutes). Wash three times with PBS, incubate for 2-15 minutes, and then wash three times with PBS.
Note: It is recommended to perform a preliminary experiment. Stain for 1 minute, rinse off the fluorescent dye, and observe the staining under a microscope. If the staining is weak, continue adding fluorescent dye to enhance the staining intensity until the desired intensity is achieved before proceeding to the next step.

4. Antibody Elution
Add an appropriate amount of completely dissolved primary and secondary antibody eluent (specially for mIHC experiments, HY-D2776) preheated at 37°C to cover the sections. Incubate at 37°C for 5-20 minutes. Discard the eluent and repeat this process. Wash three times with PBS for 5 minutes each.

5. DAPI Staining
It is recommended to repeat the staining starting from step 1.4. After staining, add DAPI working solution dropwise to the sample and incubate at room temperature for 5 minutes. Wash once with TBST and mount the slide with anti-fading mounting medium.
Note: For long-term storage, it is recommended to seal the edges of the coverslip with clear nail polish.

II. Cell Slide Staining
1. Cell Slide Sample Processing
1.1 Fix with paraformaldehyde for 15 min, wash three times with PBS for 5 min each, permeabilize with TritonX100 for 15 min, and wash three times with PBS for 5 min each.
1.2 Block endogenous peroxidase: Add 3% hydrogen peroxide solution to the wells of the cell plate, incubate at room temperature in the dark for 15 minutes, and wash three times with PBS (pH 7.4), each for 5 minutes.
1.3 BSA blocking: Add 3% BSA-PBS (or other blocking solution) to the wells of the cell plate until the cells are evenly covered. Block at room temperature for 30 minutes, then wash.
2. Antibody Incubation
2.1 Add the primary antibody, gently shake off the blocking solution, and apply the primary antibody diluted in antibody diluent to the sections. Incubate the sections flat in a light-proof humidified chamber at 4°C overnight or at 37°C for 1-2 hours. Be careful not to let the sections dry out.
2.2 Secondary antibody incubation: Wash the slides three times with PBS (pH 7.4) on a destaining shaker, each for 5 minutes. After lightly drying the sections, add a drop of secondary antibody over the tissue and incubate at room temperature in the dark for 50 minutes. Wash three times with PBS, each for 5 minutes.

3. Staining
Use a mixture of TSA dye and TSA buffer at a ratio of 1:50-1:200. Add the prepared TSA fluorescent dye reaction solution evenly over the tissue and incubate at room temperature for 1-15 minutes (optimal time is 5-10 minutes). Wash three times with PBS, incubate for 2-15 minutes, and then wash three times with PBS.
Note: It is recommended to perform a preliminary experiment. Stain for 1 minute, rinse the fluorescent dye, and then observe the staining under a microscope. If the positive staining is weak, continue adding fluorescent dye to enhance the staining intensity until the desired intensity is achieved before proceeding to the next step.

4. Antibody Elution
Add an appropriate amount of completely dissolved primary and secondary antibody eluent (specially for mIHC experiments, HY-D2776) preheated at 37°C to cover the sample. Incubate at 37°C for 5-20 minutes. Discard the eluent and repeat this process. Wash three times with PBS for 5 minutes each.

5. DAPI Staining
After staining, add DAPI working solution to the sample and incubate at room temperature for 5 minutes. Wash once with TBST and mount the slide with anti-fading mounting medium.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

Liquid

Colorless to light yellow

[Primary antibody and secondary antibody eluent, specially for mIHC experiments]

Room temperature in continental US; may vary elsewhere.

4°C, protect from light

*In solvent : -80°C, 6 months; -20°C, 1 month (protect from light)