Immunohistochemistry-Frozen

Immunohistochemistry (IHC) is a method for detecting specific antigens or proteins in tissue sections. It uses known antibodies as probes to specifically bind to specific antigens (such as peptides and proteins) in tissues or cells. Subsequently, chemical reactions are used to visualize these antigen-antibody complexes, enabling qualitative, localization, and even quantitative studies of unknown antigens in tissues or cells. The main tissue samples used in the experiments include paraffin sections and frozen sections.
The basic principle of immunoenzyme-labeled method is to first interact with the tissue with enzyme-labeled antibody, and then add the enzyme substrate to generate colored insoluble products or particles with a certain electron density. Through light or electron microscopy, various antigen components on the cell surface and inside the cell are studied. At present, peroxide-antiperoxidase (PAP) method, vitelloin-biotin-peroxidase complex (ABC) method, Streptomyces biotin-peroxidase link (SP) method are widely used in pathological diagnosis.

MCE has not independently verified the accuracy of these methods. They are for reference only.

1. Sampling

Take fresh tissue and cut it into small pieces.

2. Quick Freezing

Place the tissue pieces flat in a clean small box, add OCT embedding agent to soak the tissue, and then quickly freeze it into blocks in a liquid nitrogen or isopentane dry ice bath. If storage is required, it should be stored at -80℃ for later use.

3. Sectioning

(1) Place the cryostat in a low-temperature, sealed chamber. Place the frozen tissue sample into the cryostat, set the temperature to -20°C, and keep it overnight. Continuously cut thin sections of 5-10 µm. The thickness of the sections depends on the specific tissue; in principle, densely celled tissues should be cut thinly, while sparsely celled tissues can be cut slightly thicker.
(2) Collect the sections with a brush and place them on a glass slide for subsequent fixation.

4. Fixation

Clamp the frozen tissue block onto the cryostat holder, start the coarse advance/reverse button, and rotate the knob to smooth the tissue.
(1) Dry the frozen sample at room temperature for 15-20 minutes.
(2) Select a suitable fixative. Most low molecular weight proteins, peptides, and enzymes can be fixed with 10% neutral buffered formalin (NBF) or 4% paraformaldehyde (PFA); for large molecular weight protein antigens, 100% acetone or methanol can be used.
(3) Immerse the tissue slides in the fixative and incubate the samples at room temperature for about 15 minutes.
(4) Wash the tissue sections three times with PBST.

5. Blocking

This step prevents high background staining in the images.
(1) Wash the slides twice with PBST, 5 minutes each time.
(2) Incubate the slides in a protein blocking agent at room temperature for 30-60 minutes.
(3) Wash the slides three times with PBST, 5 minutes each time.
(4) Perform subsequent immunostaining.

6. Antibody Incubation

It can be divided into direct methods (also known as one-step methods) and indirect methods (two-step, three-step, or multi-step methods). Direct detection is a one-step process where the primary antibody directly binds to a conjugate (e.g., fluorescent dye, enzyme, colloidal gold, or biotin). Indirect methods involve conjugating a secondary antibody to indirectly stain the tissue.
6.1 Direct Method
(1) Determine the optimal antibody dilution ratio to be used and dilute the antibody in PBS containing 1% BSA.
(2) Incubate the tissue sections in the diluted primary antibody. Normally, incubate at room temperature for 1 hour or overnight at 4°C.
(3) Wash the tissue sections three times with PBST.
(4) Next, perform counterstaining, mounting, and imaging.

6.2 Indirect Method
(1) Determine the optimal antibody dilution ratio to be used. Dilute the primary and secondary antibodies with PBS containing 1% BSA.
(2) Incubate the tissue sections in the diluted primary antibody. Incubation is usually for 1 hour at room temperature or overnight at 4°C.
(3) Wash the tissue sections three times with PBST.
(4) Incubate the sample with the pre-diluted secondary antibody. Incubation for 45-60 minutes at room temperature is generally recommended.
(5) Wash the tissue sections three times with PBST.

7. Detection

Colorimetric methods, such as chemical staining or fluorescence staining, can be used.
7.1 Chemical Staining Method
(1) Immerse the slide in the chemical staining substrate solution.
(2) Wash the slide with tap water to remove excess staining solution.
(3) Wash the tissue sections with cold tap water to remove excess staining solution and make hematoxylin blue.
(4) Add a few drops of mounting medium to the slide: let the slide stand at room temperature for 5 minutes.
(5) Carefully place the coverslip onto the slide with tweezers.
(6) Image the tissue sections using a microscope. If not used immediately, store the slide at 4°C.
7.2 Fluorescent Staining Method
(1) Immerse the tissue sections in counterstaining solution (optional).
(2) Wash the slide with ice-cold deionized water to remove excess staining solution.
(3) Add a few drops of mounting medium to the slide. Let stand at room temperature for about 5 minutes.
(4) Carefully place the coverslip onto the slide with tweezers.
(5) Image the tissue sections using a microscope. If not used immediately, store the slides at 4°C, protected from light.

1. If concerned about the presence of endogenous biotin or peroxidase in the sample, endogenous avidin/biotin blocking and endogenous peroxidase blocking can be performed.
2. If using a fluorophore when incubating secondary antibodies in the indirect method, incubation must be performed in the dark.
3. During antibody incubation, the antibody solution needs to completely cover the sample.
4. If the mounting medium you are using contains a fluorescent counterstain, an additional counterstaining step is not required.