Immunohistochemistry (fluorescence) assay

Summary

After initial determination of pathological development by histologic observation, further in situ confirmation of potentially high/low expressed molecules is often required to hypothesize the molecular basis of the pathological phenotype. At this point it is necessary to use immunohistochemistry to qualitatively/semi-quantitatively examine the expression of the target protein. Sometimes, in order to determine the exact location of protein expression more precisely, it is necessary to co-stain multiple molecules using immunofluorescence co-localized with marker proteins, and to scan and overlay the images with independent channels under a confocal microscope to obtain high-resolution fluorescence co-staining results.

Operation method

Immunohistochemistry (fluorescence) assay

Materials and Instruments

Equipments: 60 ℃ temperature box, wet box, (fluorescence) body microscope
Reagents:
① Xylene
② anhydrous ethanol
③ 3% hydrogen peroxide
④ Specific primary antibody
⑤ Secondary antibody and DAB detection system
⑥ Hematoxylin

Move

The basic procedure for immunohistochemistry (fluorescence) can be divided into the following steps:
A. Soak tissue sections in xylene for 10 minutes or more, replace the xylene and soak for another 10 minutes or more.
B. Soak in anhydrous ethanol for 3-5 minutes.
C. Soak in 95% ethanol for 3-5 minutes.
D. Soak in 70% ethanol for 3-5 minutes and rinse in distilled water.
E. Incubate in 3% hydrogen peroxide for 10-30 minutes to inactivate endogenous peroxidase activity. E. Incubate with 3% hydrogen peroxide for 10-30 minutes to inactivate endogenous peroxidase activity.

F. Rinse with distilled water and soak in PBS for 5 minutes.

G. Repair with antigen: Steamer repair for 30 minutes, allow to cool naturally.
H. Serum blocking: 15-30 minutes at room temperature, consistent with the source of the secondary antibody. Discard, do not wash.
I. Appropriately diluted primary antibody is added dropwise and left overnight at 4 °C. The next day, rewarm and rinse with PBS. Rinse with PBS for 3 minutes (5 times) after rewarming the next day.
J. Incubate with biotin-labeled secondary antibody dropwise for 30 minutes at room temperature or 37 °C.
K. Incubate with PBS for 3 minutes (3 times).
L. Incubate with SP (streptavidin-peroxidase) dropwise for 30 minutes at room temperature or 37 °C.
M

. Incubate with PBS for 3 minutes (3 times)

. N. DAB color development, the degree of color development under the microscope.
O. PBS or tap water rinse for 5 minutes.
P. Hematoxylin restaining for a few moments, the nucleus can be lightly stained, and the ammonium chloride returns to the blue color.
Q. Distilled water to wash off the excess salt solution.
R. Routine dehydration, transparency, and sealing of tablets, sealing can be stored for a long time after the sealing.

Caveat

1. the use of anti-dehiscence treated slides to prevent specimens from falling off the slide during processing, especially hydrogen peroxide treatment.

2. Antigen repair commonly used repair methods include high pressure repair, microwave or steam repair, trypsin repair. We usually use 0.01 mol/L citric acid buffer of pH 6.0 to submerge the slides, and then use a steamer to repair the slides for about 30 minutes. Note that after repair, the temperature of the repair solution should be cooled naturally to room temperature.

3. Serum sealing The sealing serum is usually from the same species as the secondary antibody, e.g., goat serum sealing solution, or calf serum, BSA, etc., but not from the same source as the primary antibody. Generally 10-30 minutes at room temperature, to prevent over-containment affecting the binding of primary antibody.

4. Primary and secondary antibody concentration and incubation time Primary antibody incubation conditions are the most important in the immunohistochemical reaction, including incubation time, temperature and antibody concentration, which need to be explored according to the specific situation, generally according to the starting concentration provided by the antibody instruction manual and adjusted. There are several incubation temperatures for primary antibody: 4 ℃, room temperature, 37 ℃, of which 4 ℃ has the best effect; incubation time: this is related to the temperature and antibody concentration, generally 4 ℃ overnight. Incubation conditions for secondary antibody: Incubate the secondary antibody at room temperature or 37 ℃ for 30-60 minutes, and the specific time needs to be explored.

5. The depth of DAB background and specific staining can be determined by DAB incubation conditions; the time of DAB incubation is not fixed, but is mainly controlled by the microscope, and can be washed out when the specific staining is strong and the background coloring is light.

6. For long term storage, we generally use neutral gum to seal the slides, and cover the coverslips slowly from one side to avoid air bubbles. An example of the results is shown in Figure 8-2-5.Immunofluorescence is similar to immunohistochemistry in that frozen sections are used and do not require antigen repair or inactivation of endogenous peroxidase. Two or more primary antibodies of different genera are used, differentiated by different fluorescently labeled secondary antibodies, appropriate nuclear markers such as DAPI are added to show tissue morphology, and the sections are blocked with 15% to 30% glycerol/PBS. The finished sections can be stored at 4 ℃ for 1 week, but it is better to observe and take pictures within the shortest time to maintain the integrity of the tissue morphology. Examples of results are shown in Figure 8-2-6.

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