Absin 2-Color IHC Kit (Anti-Rabbit and Mouse Secondary Antibody)

1 Quenching, transparency, sealing

a ) Removing the frozen glass slides from the refrigerator and restoring them to room temperature, PBST immersion 3min , repeat 3 Secondary

b ） 10% Neutral formalin or 4% paraformaldehyde dip tablets 10-30min Sterilized water washing tablets 1min , repeat 3 Twice. (Optional, depending on sample quality)

c ) Dropwise adding membrane breaking agent to permeate 15min ， PBST immersion 3min , repeat 3 Twice. (Optional, generally not required)

d ) Circle the sample area on the slide with a histochemical pen, add peroxidase blocking solution dropwise to cover the sample area, and incubate 10min ， PBST immersion 3min , repeat 3 Twice.

e ) Remove that residual washing solution on the slide, add the blocking solution dropwise, cover the sample area, and incubate at room temperature 60min The blocking solution was removed.

2 Primary antibody incubation

a ) Remove the closure on the slide.

b ) The diluted primary antibody solution was added dropwise with a pipette to immerse the sample area.

c ) Room temperature moisturizing shaking incubation 1h (need to be optimized for different antibodies), or 4℃ Incubate overnight.

d ) with 1×TBST buffer Immersion slide 3min , repeat 3 Twice.

3 Secondary antibody incubation

a ) Remove any remaining lotion from the slide.

b ) direct dropwise addition HRP Secondary antibody working solution, immerse the sample area.

c ) Moisturizing incubation at room temperature 10min 。

d ) with 1×TBST buffer Immersion slide 3min , repeat 1 Twice.

4 Fluorescence staining to amplify the signal

a ) Remove any remaining lotion from the slide.

b ) drop-on slide with pipette 1× Dye working solution 50-100ul (Use signal amplifier to press 1:200 Dilute) to immerse the sample area.

c ) Room temperature moisturizing shaking incubation 10min 。

d ） 1×TBST buffer Dip slides, room temperature dip slides 3min 。 repeat 3 Twice.

e ) dropwise addition of antibody eluate ( abs994 ）， 37℃ incubation 20-30min 。

f ) Sterilized water washing tablets 1 times, 1×TBST buffer Immersion tablet 2min 。

5 A new round of dyeing (single dyeing can be directly performed on the first 6 Step)

a ) After each round of staining, fluorescence microscope can be used to confirm the staining. TBST Cover the sample to prevent dry sheets.

b ) Repeat steps 2-4 。

c ) Multiple rounds of staining repeat steps 5 Nucleation and sealing were performed after completion.

6 Nucleation and sealing

Dropwise 1×DAPI Working solution onto the sample, immerse the sample area, and incubate at room temperature 10min 。 use 1×TBST Immersion slide 3 Time, each time 2min 。 Then add antifluorescent quenching sealant dropwise, seal with cover glass, avoid bubbles, long-term storage, please use transparent nail polish to seal the edge of the cover glass.

7 The stained tissue slices were observed and analyzed under a fluorescence microscope.

V. Description of results:

1 Changes in microwave repair antigen, incubation time and temperature may lead to wrong results.

2 In each staining process, there must be tissue positive control and reagent negative control experiments simultaneously.

3 If the positive tissue control cannot show positive staining, the test result of this batch of samples shall be judged to be invalid.

VI. Product performance indicators:

1 Conformity: Take the conforming tissue slices (including the positive tissue slices and the negative reagent control), and after the corresponding immunohistochemical test, the staining result of the positive control is positive, and the staining result of the negative control is negative.

2 Intra-batch repeatability: Take the same batch of kits and detect the same tissue section 3 Tablet, the staining results should be consistent.
Multiplex fluorescence immunohistochemistry ( mIHC ) Experimental operation demonstration video
VII. Dye Information Form 

8. Other product recommendations

There are complex cellular composition in tissue microenvironment, and the phenotype, state, abundance and distribution of these cells have important biological significance and clinical value. It can be presented in situ in tissue with the help of antibody staining. Immunohistochemical staining is a common technique to study tissue morphology and in situ protein expression. Conventional IHC detection can only display a single indicator, and it is difficult to present the cell composition, state and relationship in the complex tissue microenvironment, and this information is crucial for the diagnosis and treatment of diseases!
Principle of tyrosine signal amplification technology: Similar to the DAB color development method of conventional immunohistochemistry, TSA technology also uses HRP-labeled secondary antibody, and HRP catalyzes the addition of fluorescein substrate in the system to produce an activated fluorescent substrate, which can be covalently bound with tyrosine on the antigen, so that fluorescein is stable and covalently bound on the sample. Thereafter, the non-covalently bound antibody was washed off by thermal repair method, and a primary antibody was replaced for a second round of incubation, and another fluorescein substrate was replaced, so that multiple labeling can be achieved by reciprocating.

TSA Monochromatic fluorescent dye 570 Signal amplification reaction solution, anti-pika HRP Labeled secondary antibody, anti-fluorescence quenching tablet sealing agent, DAPI Peroxidase blocking solution

Fluorescent dyes should be kept away from light, stored at 2-8℃, and the validity period is 12 months.