Ready-to-use SABC immunohistochemical staining kit

user's Guide

Product Numbers: kit09-2—Rabbit IgG

Reagent storage: It can be stored at 4 ℃ for one year, and freezing should be avoided.

working principle

SABC is specifically designed for immunohistochemistry and other immunoassays to show the distribution of antigens in tissues and cells. Streptavidin is a protein extracted from Streptomyces and has a molecular weight of 47,000. Like avidin, it has a very high affinity for biotin molecules, which is one million times the affinity of general antigens and antibodies. Avidin is a basic protein (IP = 10), which can be transformed into a neutral protein after modification. The isoelectric point of streptavidin is close to neutral, IP = 6.0 ~ 6.5, the non-specific adsorption to tissues and cells is very low, and the background of immunohistochemical methods based on streptavidin is very low. SABC stands for StreptAvidin-Biotin Complex. According to research, SABC can form a complex of about one hundred peroxidases and about fifty streptavidins. A large number of enzymes will ensure that SABC has a high sensitivity. SABC has the advantages of high sensitivity, low background and easy operation.

Application range

It is suitable for immunohistochemical methods to display the distribution of the antigen to be tested. The kit is suitable for conventional paraffin embedded sections, frozen sections, cultured fixed cell sections, and freshly prepared blood smears, slides, etc.

What's in the kit

1. Normal rabbit serum blocking solution: 10ml. Used for sealing of tissue sections.

2. Secondary antibody: 10ml. Affinity purified antibody, labeled long arm biotin. Biotin rabbit anti-goat IgG.

3. SABC: 10ml. Streptavidin-peroxidase complex.

User-supplied reagents:

1. Sticky tablets APES or POLY-L-LYSINE.

2. 0.02 M PBS (pH7.2-7.6) mixing method: add 8.5g of sodium chloride, 2.8g of Na2HPO4 and 0.4g of NaH2PO4 in 1000ml of distilled water. If water-containing phosphate is used, the water content in the molecular formula should be added. 0.01M citrate buffer: 1000ml of distilled water, add 3g of trisodium citrate (C6H5Na3O7 · 2H2O), 0.4g of citrate (C6H8O7 · H2O).

3. DAB color development kit.

Selection of immunohistochemical staining program: The user needs to determine the program according to the characteristics of the antigen / antibody. In most cases, the staining results of each program must be compared first.

Procedure A: Paraffin section hot repair antigen procedure. Suitable for nuclear antigens, antigens that are easily destroyed by fixation.

Procedure B: Paraffin section enzyme digestion procedure. It is suitable for antigens that are fixed and shielded.

Procedure C: Paraffin section non-digestion / non-repair procedure. Suitable for stable antigens.

Procedure D: Cell smear, frozen section staining procedure.

A. Microwave repair antigen staining procedure for paraffin sections:

1. Glass slide anti-off tablet treatment: choose APES or Poly-Lysine. After removing the slices, place them in the oven at 58-60 ℃ for 30-60 minutes to make the slices adhere tightly.

2. The slices are routinely dewaxed to water.

3. Mix 1 part of 30% H2O2 + 10 parts of distilled water at room temperature for 5-10 minutes to inactivate endogenous enzymes. Wash 3 times with distilled water.

4. Heat repair antigen: Immerse the slice in 0.01M citrate buffer solution (PH6.0), heat it in an electric stove or microwave oven, and then turn off the power. After 5-10 minutes interval, repeat 1-2 times. After cooling, PBS (pH7.2-7.6) was washed 1-2 times.

4. Add 5% BSA blocking solution at room temperature for 20 minutes. Shake off excess liquid, do not wash.

5. Appropriately diluted primary antibody (goat IgG or rat), about 1 hour at 37 ℃ or about 2 hours at 20 ℃. Can also be overnight at 4 ℃. Wash with PBS (pH7.2-7.6) for 2 minutes × 3 times. (The dilution, incubation time and temperature of the primary antibody are directly related to the staining intensity and background. Generally speaking, when the positive staining intensity is not enough, the primary antibody concentration can be increased and the incubation time can be increased; when the background is too high, the primary antibody concentration can be reduced And shorten the incubation time.)

6. Add biotinylated rabbit anti-goat IgG (or rabbit anti-rat gG) at 20-37 ℃ for 20 minutes. Wash with PBS (pH7.2-7.6) for 2 minutes × 3 times.

7. The reagent SABC was added dropwise at 20-37 ° C for 20 minutes. Wash in PBS (pH7.2-7.6) for 5 minutes × 4 times.

8. DAB color development: use DAB color development kit. Take 1ml of distilled water, add 1 drop of A, B, and C reagents in the kit, mix well and add to the slice. Color development at room temperature, control reaction time under the microscope, generally between 5-30 minutes. It can also be developed by self-developing agent. Wash with distilled water.

10. Hematoxylin light counterstain. Dehydrated, transparent, sealed. Microscopic observation.

B. Paraffin section enzyme digestion procedure:

The following steps replace step 4 of the A program: dropwise add the compound digestive fluid (available from Dr. Duck) for 5-10 minutes. Wash 3 times with distilled water. It is also possible to use 0.1% trypsin as digestive juice.

C. Paraffin section enzyme indigestion / repair procedure:

For antigens that do not require microwave repair or digestion, omit step 4 of the A procedure.

D. Blood smear, cell and frozen section staining procedures

1. The slides are treated with anti-dropping tablets (Poly-L-Lysine). The anticoagulated blood is smeared after stratified centrifugation; cultured cells can also be smeared or patch grown; frozen sections are blown dry at room temperature with a fan.

2. The preferred fixation plan is 4% paraformaldehyde or 10% formalin fixation for 60-90 minutes.

3. Mix 1 part of 30% H2O2 + 50 parts of pure methanol, soak for 30 minutes at room temperature to inactivate endogenous peroxidase. Wash with distilled water 1-2 times.

4. Heat repair antigen: Immerse the slice in 0.01M citrate buffer (PH6.0), heat it in an electric stove or microwave oven to boiling, and then cut off the power. After cooling, PBS (pH7.2-7.6) was washed 1-2 times.

The remaining steps are the same as those of paraffin section 5-10.

Precautions:

1) If the staining background is too high, after the SABC reaction and before DAB color development, wash the slices 4 times with PBS (pH7.2-7.6) supplemented with 0.01-0.02% TWEEN20, 2 times with PBS alone, and then DAB color development .

Warning: DAB is a suspicious carcinogen, please take necessary precautions.

2) 0.01M citrate buffer solution (PH6.0) can be used for heat repair antigen; various buffer solutions such as PBS and TBS can also be used.

3) Deparaffinization is not complete, and non-specific background staining is prone to occur. It is recommended that the dewaxing of immunohistochemical sections be separated from conventional HE dewaxing.

4) If this kit is used for tissue sections rich in endogenous biotin, such as liver and kidney, special sealing treatment is required.

5) The activity of one or more reagents in the kits beyond the expiration date may be reduced. Therefore, expired kits must not be used, and kits between different batches cannot be mixed.