Vari Fluor 6-colour Multiple Fluorescent Staining Kit ((For mouse samples) 

Multiplex immunohistochemical, also known as Tyramide dignal amplification (TSA), is an enzymatic assay that uses horseradish peroxidase (HRP) for high-density in situ labelling of target proteins or nucleic acids. It is a class of enzymatic assays that use horseradish peroxidase (HRP) for high-density in situ labelling of target proteins or nucleic acids. It is based on multiple cis-immunostaining with tyramide signal amplification, which allows the detection of multiple targets in situ in cell or tissue samples, and elucidation of their interaction mechanisms through the study of their combinations and positional relationships.

MCE offers a wide range of multiplex fluorescence kits for simultaneous detection of up to six targets to maximise customer satisfaction.

4 ºC, 1 years

Avoid repeated freeze-thaw cycless

Dye Preparation

working solution = VF fluorescent dye + TSA buffer; the recommended dilution ratio of fluorescent dye and TSA buffer is 1:200; the dilution ratio can be adjusted and optimised flexibly according to the specific situation, and the best range is 1:50--1:400; it is generally recommended that the dilution ratio is 1:50--1:200 if the primary antibody incubation time is within 1h--3h at room temperature; 1:200 if the primary antibody incubation time is 4 hours overnight (12h or more), and 1:200--1:400 or higher. 1:200, if the primary antibody incubation time is 4 hours overnight (12h or more), it is recommended that the dilution ratio is 1:200-1:400 or higher.

Detailed operation steps

1 Sample preparation

1) Paraffin sections: sequentially put the sections into xylene1, 15min-xylene, 15min-anhydrous ethanol, 5min-anhydrous ethanol, 5min-95% ethanol 5min-85% ethanol 5min-75% ethanol 5min, and wash with distilled water.

2) Frozen sections: frozen sections were fixed for 10-30min, washed with PBS for 5min, repeated 3 times, permeabilised with 0.3% triton-X100 membrane-breaking solution for 20min, washed with PBS for 5min, repeated 3 times.

3) Cell crawler or cell smear: cell samples were fixed for 10-30min, washed with PBS for 5min and repeated 3 times, permeabilised with 0.3% triton-X100 membrane-breaking solution for 20min, washed with PBS for 5min and repeated 3 times.

Note: 1) The number of cells contained in the tissue should be greater than 1000.

2) The thickness of the section is recommended to be about 4 μm, using anti-dehiscence slides, and it is recommended that the slides be prepared within one week after fixation.

3) Formalin-fixed wax blocks or slides, large pieces of tissue or TMA, sealing wax should not have obvious breaks.

4) Tissues should be fixed in 10 % neutral formalin for a normal fixation time of 18-24 h. The tissue should be fixed in 10 % neutral formalin.

5) For slide samples, the tissue needs to fit snugly on the slide to avoid wrinkles, and the slide should not be broken, scratched or stained.

2 Antigen Retrieval

2.1 Mode

1) Citric acid Retrieval 1) Boil the slide in pH 6.0 , 10 mM sodium citrate buffer using a microwave oven.

2) Hold at low fire temperature for 10 min (take care to replenish the solution).

3) Place the slide on the bench and cool to room temperature for 30 min.

2.2 Mode 2: EDTA Retrieval

1) Use a microwave oven and boil the slide in 1 mM EDTA, pH 8.0.

2) Keep it under low heat for 15 min without cooling. 3 Blocking endogenous peroxidase

1) Wash the sections three times with dH₂O for 5 min each time.

2) Place the sections in 3 % hydrogen peroxide solution and incubate for 10 min.

3) Wash the sections twice with dH₂O for 5 min each time.

4) Wash with TBST for 5 min. 4 Non-specific target closure

1) Remove residual wash from the slide.

2) Circle the area of the sample on the slide with a histochemical pen, add a drop of closure solution and submerge the sample.

3) Humidify and shake for 10 min at room temperature.

5 Primary antibody incubation

1) Remove the residual liquid on the slide.

2) Add the primary antibody working solution dropwise with a pipette gun and submerge the sample area.

3) Incubate for 1 h at room temperature with humidified shaking. Note: The incubation time is adjusted according to different antibodies, and avoid drying by operating in a wet box. 4) Wash with TBST for 3 min and repeat once.

6 HRP secondary antibody incubation

1) Remove the residual liquid on the slide.

2) Add HRP secondary antibody working solution dropwise with a pipette gun and submerge the sample.

3) Incubate for 10 min at room temperature with shaking.

4) Wash with TBST for 3 min and repeat.

7 TSA Fluorescent Dye Reaction Solution Reaction.

1) Remove the residual liquid from the slide.

2) Prepare a working solution by diluting the TSA dye at 1 : 200 with 1× Tyramide Amplification Buffer. Prepare 100 μL of staining solution for each sample.

Note: The staining solution can be stored at room temperature, protected from light, for a maximum of 24 hours.