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Absin 7-Color IHC Kit (Anti-Rabbit and Mouse Secondary Antibody)

Absin 7-Color IHC Kit (Anti-Rabbit and Mouse Secondary Antibody)

Catalog Number: abs50037 Brand: Absin
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$710 USD
$710 USD
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Product Specification

References 1. Mingzhu Yang, et al., Chemical-induced chromatin remodeling reprograms mouse ESCs to totipotent-like stem cells. Cell Stem Cell 2022 Mar 3 Xin Guan, et al., Nanoparticle-enhanced radiotherapy synergizes with PD-L1 blockade to limit post-surgical cancer recurrence and metastasis. Nature Communication. May 20, 2022 Qian-Yue Zhang, et al., Lymphocyte infiltration and thyrocyte destruction are driven by stromal and immune cell components in Hashimoto's thyroiditis. Nature Communications Feb 9, 2022 4. Dabbs David J. Diagnostic immunohistochemistry (M). Beijing: Peking University Medical Press, September 2008. Ed Harlow, David Lane. Technical guidelines for antibodies (M). Beijing: Science Press, 2002: 79-80, 105. Stack, E. C., et al., Multiplexed immunohistochemistry, imaging, and quantitation: a review, with an assessment of Tyramide signal amplification, multispectral imaging and multiplex analysis. Methods, 2014 70 (1): 46-58. 7. Money helps the country. Jiao Lei. Application of multi-labeled immunofluorescence staining and Doppler imaging in histological studies. Chinese Journal of Histochemistry and Cytochemistry, 2017 (4): 373-382.
Usage I. Specimen requirements
1. Formalin-fixed paraffin blocks or slides, large tissue sections or TMAs; paraffin seal must be intact.
2. For slide specimens, tissue must adhere tightly to the slide without folds; slides must be free of cracks, scratches or stains.
3. Tissue must contain >1 000 cells.
4. Blocks must contain solid tumour tissue; necrotic tumour, fine-needle aspirates or cytospins will impair staining.
5. Tissue must be fixed in 10 % neutral-buffered formalin for 18–24 h.
6. Cut 4 µm sections on adhesive slides; prepare slides within one week after fixation.
7. Do NOT use any adhesives in the water-bath; place tissue on the front centre of the slide. Drain vertically on absorbent paper and gently tap off droplets—never wipe the slide.
8. Dry slides on a 45 °C hot plate for 30 min (air-dry ≥1 h if no hot plate is used).

II. Assay procedure
1. Equipment required: pipettes, oven, microwave, IHC pen, antigen-retrieval vessel, staining jars, timer, humid incubation box, coverslips, fume hood, wash bottle, fluorescence microscope, 100 mL & 1 000 mL cylinders, etc.
2. Reagents required: sterile de-ionised water (abs9259), xylene, ethanol (100 %, 95 %, 70 %), 10 % neutral-buffered formalin, primary antibodies, blocking buffer, TBST, etc.
3. Reagent preparation
1) Fluorophore dilution: 100× stock dyes are diluted 1:100 in signal-amplification buffer (prepare fresh). DAPI 100× stock is diluted 1:100 in sterile water.
2) Secondary antibody: the kit supplies a mouse/rabbit universal secondary; verify species compatibility (goat host).
4. Imaging: fluorescence microscope or whole-slide scanner equipped with filter sets matching the excitation/emission spectra listed in the dye table.

III. Paraffin-section protocol
1. Deparaffinisation & rehydration
a) Fresh xylene 10 min ×3.
b) Ethanol gradient: 100 % 5 min → 95 % 5 min → 70 % 2 min.
c) Sterile water rinse 1 min ×3.
d) Optional post-fixation: 10 % neutral formalin or 4 % PFA 10–30 min, water rinse 1 min ×3.
e) Optional permeabilisation: permeabilisation reagent 15 min, PBST 3 min ×3.

2. Heat-induced epitope retrieval (HIER)
a) Immerse slides in 1× antigen-retrieval buffer in microwave vessel.
b) Microwave at high power until boiling.
c) Maintain low power 15 min (top up liquid to prevent drying).
d) Cool to room temperature (RT).

3. Quenching & blocking
a) Remove excess buffer.
b) Circle tissue with IHC pen; cover with 3 % H₂O₂ 10 min, PBST 3 min ×3.
c) Block with blocking buffer 10–30 min at RT with shaking; discard.

4. Primary antibody incubation
a) Remove blocking buffer.
b) Add diluted primary antibody to cover tissue.
c) Incubate 1 h at RT with shaking (optimise if necessary).
d) 1×TBST 3 min ×2.

5. Secondary antibody incubation
a) Remove wash buffer.
b) Add HRP-conjugated secondary to cover tissue.
c) Incubate 10 min at RT in humid box.
d) 1×TBST 3 min ×2.

6. Fluorescent signal amplification
a) Remove wash buffer.
b) Add 100 µL 1× TSA fluorophore working solution (1:100 dilution) to cover tissue.
c) Incubate 10 min at RT with shaking.
d) 1×TBST 3 min ×3.
e) Microwave retrieval; cool to RT.
f) Sterile water rinse, then 1×TBST 2 min.

7. Next staining cycle (skip for single colour)
a) Check staining under microscope; keep tissue wet with TBST.
b) Block 10–30 min (no quenching needed).
c) Repeat steps 4–6.
d) After final cycle proceed to nuclear staining & mounting.

8. Nuclear staining & mounting
Add 1×DAPI working solution 5 min at RT. 1×TBST 2 min ×3. Apply antifade mounting medium and coverslip; seal edges with clear nail polish for long-term storage.

9. Imaging & analysis: visualise on fluorescence microscope.

IV. Fresh-frozen-section protocol (requires antibody-stripping buffer abs994)
1. Quenching, permeabilisation & blocking
a) Equilibrate frozen slides to RT; PBST 3 min ×3.
b) Optional fixation: 10 % neutral formalin or 4 % PFA 10–30 min, water rinse 1 min ×3.
c) Optional permeabilisation: permeabilisation reagent 15 min, PBST 3 min ×3.
d) Circle tissue; 3 % H₂O₂ 10 min, PBST 3 min ×3.
e) Block 10–30 min; discard.

2. Primary antibody: same as paraffin step 4.
3. Secondary antibody: same as paraffin step 5.
4. TSA amplification
a–d) identical to paraffin step 6a–d.
e) Add antibody-stripping buffer (abs994) 15–20 min at 37 °C.
f) Water rinse, then 1×TBST 2 min.

5. Next cycle (skip for single colour)
a–d) same logic as paraffin step 7.
6. Nuclear staining & mounting: identical to paraffin step 8.
7. Imaging & analysis.

V. Interpretation
1. Any change in retrieval, incubation time or temperature can produce erroneous results.
2. Each run must include a tissue-positive control and a reagent-negative control.
3. If the positive control fails, the entire batch is invalid.

VI. Performance characteristics
1. Accuracy: positive control tissue must stain positively; negative control must remain unstained.
2. Within-batch reproducibility: triplicate sections stained with the same kit lot must yield identical patterns.

VII. Spectral data
Fluorophore Peak wavelength (nm) Spectrally equivalent commercial dyes Suggested pseudo-colour
Ex Em
DAPI 360 461 DAPI  
TSA-480 450 480 Opal 480 / Aqua  
TSA-520 490 520 FITC / AF 488 / Opal 520  
TSA-540 520 540 AF 514 / Opal 540  
TSA-570 550 570 Cy3 / AF 555 / Opal 570  
TSA-620 590 620 AF 594 / Opal 620 / Texas Red  
TSA-650 630 650 Cy5 / AF 610 / Opal 650  
TSA-690 640 670 AF 647 / Cy5 / Opal 670  
TSA-700 680 702 AF 680 / Cy5.5 / Opal 690  
TSA-770 740 780 Cy7 / Opal 780  
VIII. Associated reagents (available at www.absin.net)
Category Cat. No. Product Size
Fixation abs9179 4 % paraformaldehyde (universal fixative) 500 mL
Embedding (frozen) abs9756 OCT compound 110 mL
Antigen retrieval (paraffin) abs9342 Tris-EDTA buffer (10×, pH 9.0) 100 mL
abs9248 Sodium citrate buffer (50×) 100 mL
Antibody stripping abs994 mIHC antibody stripping buffer 30 mL
Permeabilisation abs9149 Triton X-100 100 mL
Peroxidase quenching abs9333 H₂O₂ blocking solution 100 mL
abs9152 Tween-20 500 mL / 2.5 L
Blocking abs933 Goat serum 500 mL
abs9157 Bovine serum albumin 100 g
Primary antibodies abs1 Validated IHC antibodies -
abs9299 Antibody diluent 100 mL
Multiplex kits
(TSA dyes, DAPI, mounting medium, secondary, amplification buffer)		 abs50165 7-colour mIHC kit (770-enhanced, anti-rabbit HRP) 20/50/100 rxn
abs50166 7-colour mIHC kit (770-enhanced, mouse/rabbit universal) 20/50/100 rxn
abs50015 7-colour mIHC kit (mouse/rabbit universal) 20/50/100 rxn
abs50037 7-colour mIHC kit plus (mouse/rabbit universal) 20/50/100 rxn
abs50031 7-colour mIHC kit (anti-rabbit) 20/50/100 rxn
abs50038 7-colour mIHC kit plus (anti-rabbit) 20/50/100 rxn
abs50014 6-colour mIHC kit (mouse/rabbit universal) 20/50/100 rxn
abs50049 6-colour mIHC kit plus (mouse/rabbit universal) 20/50/100 rxn
abs50030 6-colour mIHC kit (anti-rabbit) 20/50/100 rxn
abs50048 6-colour mIHC kit plus (anti-rabbit) 20/50/100 rxn
abs50013 5-colour mIHC kit (mouse/rabbit universal) 20/50/100 rxn
abs50029 5-colour mIHC kit (anti-rabbit) 20/50/100 rxn
abs50012 4-colour mIHC kit (mouse/rabbit universal) 20/50/100 rxn
abs50028 4-colour mIHC kit (anti-rabbit) 20/50/100 rxn
abs50089 3-colour mIHC kit (mouse/rabbit universal) 100 rxn
abs50088 3-colour mIHC kit (anti-rabbit) 100 rxn
abs50087 2-colour mIHC kit (mouse/rabbit universal) 100 rxn
abs50086 2-colour mIHC kit (anti-rabbit) 100 rxn
Lung-cancer panels
(primary antibodies included)		 abs50083 Lung TME panel I 20 rxn
abs50084 Lung TME panel II 20 rxn
Buffers abs952 TBST (10×) 500 mL ×2
abs9340 PBST (1×, pH 7.4) 500 mL
abs962 PBS (1×) 500 mL
Theory Multiplex fluorescence immunohistochemical staining is based on the principle of specific binding of antigen and antibody. The secondary antibody labeled with horseradish peroxidase (HRP) is selected to activate the fluorescent dye in the kit and covalently bind the signal to the antigen. In situ multi-target staining of tissues or cells is achieved by multiple rounds of staining cycles with the aid of different fluorescent dye labels.
Synonym Multicolor kit
Description

The tissue microenvironment harbors a complex cellular composition whose phenotype, state, abundance, and spatial distribution carry profound biological significance and clinical value. These characteristics can be visualized in situ by antibody-based staining. Immunohistochemistry (IHC) is a routine technique for interrogating tissue architecture and in-situ protein expression; however, conventional single-color IHC reveals only one marker at a time, making it insufficient to depict the intricate cellular constituents, states, and inter-cellular relationships within the microenvironment—information that is indispensable for disease diagnosis and therapy.

Tyramide signal amplification (TSA) principle: analogous to the DAB chromogenic method used in standard IHC, TSA employs an HRP-conjugated secondary antibody. Horseradish peroxidase catalyzes the added fluorogenic tyramide substrate, generating highly reactive fluorescent tyramide radicals that covalently bind to tyrosine residues on nearby proteins, stably depositing fluorophore at the antigen site. Subsequent heat-mediated stripping removes non-covalently bound antibodies, allowing sequential rounds of primary antibody incubation with a different fluorophore-conjugated tyramide substrate. Iteration of this cycle enables high-plex multiplexed labeling.
Composition
Pack size abs50037 (mouse/rabbit) 7-color multiplex fluorescent IHC kit (plus)
Component TSA-480 TSA-520 TSA-570 TSA-620 TSA-690 TSA-770 DAPI 100× Signal amplification buffer Poly-HRP-conjugated secondary antibody (goat, mouse/rabbit universal) Antifade mounting medium
100 reactions Volume 100 µL 100 µL 100 µL 100 µL 100 µL 100 µL 100 µL 60 mL 60 mL 5 mL × 2
50 reactions 50 µL 50 µL 50 µL 50 µL 50 µL 50 µL 100 µL 30 mL 30 mL 5 mL
20 reactions 20 µL 20 µL 20 µL 20 µL 20 µL 20 µL 100 µL 12 mL 12 mL 5 mL
Kit components: TSA monomeric fluorophore 480, TSA monomeric fluorophore 520, TSA monomeric fluorophore 570, TSA monomeric fluorophore 620, TSA monomeric fluorophore 690, TSA monomeric fluorophore 770, signal amplification buffer, mouse/rabbit universal HRP-conjugated secondary antibody, antifade mounting medium, DAPI.
General Notes 1. This kit is intended exclusively for immunohistochemistry; any other application is prohibited.
2. Operation must be performed only by qualified laboratory personnel.
3. Appropriate personal protective equipment must be worn to prevent contact with skin and eyes.
4. Reagents may lose activity after the stated expiry date; do not use beyond expiration.
5. Mixing any staining component of this kit with products from other manufacturers may lead to unpredictable staining artefacts.
6. Incomplete de-paraffinisation can compromise staining quality.
  1. To avoid false-negative or false-positive results, always run a positive tissue control and a negative reagent control in parallel.
  2. All waste generated during use must be disposed of in accordance with the Regulations on the Administration of Medical Waste.
Storage Temp. Fluorescent dyes should be stored in the dark from light at 2 ~ 8 ℃, and the validity period is 12 months from receipt of the goods.
Applications It is mainly used for immunohistochemical staining of tissues, paraffin sections and TMA chips. It can also be used for frozen sections and cell crawling sections, and needs to be matched with abs994 antibody eluate (specific for mIHC).

Picture

Immunohistochemistry