Schematic diagram of frozen section/cell crawling staining process:

Protocol for frozen section：

1. Preparation of frozen section: Take the frozen section out of the refrigerator and let it dry at room temperature. Fix it for 10-30min after drying. Wash it for 5min with PBS and repeat 3 times.

2. Slicing and Membrane Breakage: Add the membrane-breaking solution and permeate for 20 minutes. Wash with PBS for 5 minutes, repeat 3 times. (This step applies to intracellular indicators; skip if not applicable)

3. Antigen Retrieval: Place the slide in a retrieval chamber filled with EDTA antigen retrieval buffer (pH 9.0) and incubate at 60°C for 30 minutes in a water bath. Allow to cool naturally, then wash with PBS for 5 minutes, repeat 3 times (optional step as retrieval helps expose antigens and remove endogenous peroxidase).

4. Blocking endogenous peroxidase: the section was placed in a 3% hydrogen peroxide solution and incubated at room temperature in the dark for 25 min. The glass slide was placed in PBS (PH7.4) and shaken in a decolorization shaker for 3 times, each time for 5 min.

5. BSA blocking: after slightly drying the section, draw a circle around the tissue with a histological pen (to prevent antibody flow away), and add 3% BSA (or other blocking solution) to the circle to cover the tissue evenly, and block at room temperature for 30min.

6. Primary Antibody: Gently shake off the blocking solution and add the primary antibody diluted in antibody dilution buffer to the slide. Place the slide flat in a light-shielding humid box and incubate overnight at 4°C or 1-2 hours at 37°C (add a small amount of water to the humid box to prevent antibody evaporation).

7. HRP Secondary Antibody: Place the slide in PBS (pH 7.4) and wash three

times with decolorization shaker, each for 5 minutes. After gently drying

the slide, add the HRP secondary antibody corresponding to the species

of the primary antibody into the ring-shaped area to coat the tissue.

Incubate at room temperature under light protection for 50 minutes. Wash

three times with PBS, each for 5 minutes.

8. Fluorescent dye reaction: Mix concentrated fluorescent dye with TSA buffer at a 1:50-1:200 ratio thoroughly. Apply the prepared TSA fluorescent dye reaction solution uniformly to tissue sections. React at room temperature for 1-15 minutes (optimal duration: 5-10 minutes). Wash with PBS three times. (For preliminary experiments: After staining for 1 minute, rinse off the fluorescent dye under microscope to observe staining intensity. If the fluorescence is weak, continue adding fluorescent dye to enhance intensity until satisfactory results are achieved before proceeding to the next step.) React for 2-15 minutes. Wash with PBS three times.

9. Last antibody elution: Add an appropriate amount of antibody elution solution preheated to 37 degrees Celsius until completely dissolved to cover the sample, place it at 37 degrees Celsius for 5-20 minutes, discard the elution solution, add an appropriate amount of antibody elution solution to cover the sample again, place it at 37 degrees Celsius for 5-20 minutes, discard the elution solution, PBS wash three times, each time 5 minutes.

10. Repeat steps 4-9 (using a different fluorescent dye) ---Second round of labeling

11. Repeat steps 4-8 (using a different fluorescent dye) ---Third round of labeling

             ............................ ---Nth round of labeling

12. DAPI staining of nuclear: Place the slide in PBS (PH7.4) and shake it on a decoloring shaker for 3 times, each time for 5min. After slightly drying the section, add DAPI staining solution in a ring and incubate at room temperature in the dark for 10min.

13. Mounting: The glass slide was placed in PBS (PH7.4) and shaken on the decolorization shaker for 3 times with washing, each time for 5min. After slight drying of the section, the anti-fluorescence quenching mounting agent was used for mounting.

14. Microscopic photography: The section is observed and images are collected under the fluorescence microscope/cooperative focus/multichannel fluorescence scanner/multispectral imaging system.

List of kit products:

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For more information please contact us info@neorise.net.

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