Follow the straightforward steps under to pick out essentially the most acceptable labeling and detection methodology in your experiment.
| 1. Select the first antibody ⯈ Specific to your antigen ⯈ Consider the kind of tissue and its preparation (fixation) ⯈ Consider antigen unmasking requirementsVECTABOND reagent (tissue part adhesive) Blocking brokers ⯈ Options decided by the variants chosen in steps 1-2 ⯈ Streptavidin / biotin blocking package ⯈ (if you’re utilizing a biotin / streptavidin system) ⯈ Avidin / biotin blocking package ⯈ Normal serum (from secondary antibody sort) ⯈ MOM (Mouse to Mouse) blocking agent ⯈ RTU Animal-Free blocking agent and diluent ⯈ BSA ⯈ Casein answer |
| 2. Select secondary antibody and tertiary detection system ⯈ Select the fluorophore primarily based on the wavelengths of the filters within the microscope ⯈ Fluorophore-conjugated secondary antibody or biotinylated secondary antibody ⯈ Consider sensitivity necessities ⯈ Consider the kind of main antibody ⯈ Consider the kind of tissue Variant A One step ⯈ One-mark. Fast. Conveniently. ⯈ Fluorophore-conjugated secondary antibodies ⯈ VectaFluor RTU DyLight labeled secondary antibodies Option B One step Dual-labeling, detection of two antigens. Fast. Conveniently. ⯈ VectaFluor Duet IF kits for double marking Variant C Two steps ⯈ Based on biotin. ⯈ Biotinylated secondary antibody and fluorophore conjugated to avidin or streptavidin. ⯈ See step Three for extra amplification Option D Two steps ⯈ Highest sensitivity. It just isn’t primarily based on biotin. ⯈ VectaFluor Excel Amplified fluorescent staining system (amplification linker + fluorescent tertiary antibody) |
| 3. Select sign amplification utilizing Biotin -based methods (Step 2, variant C) VÃce With biotin-based methods, a number of amplification steps might be carried out  |
| 4. Reduction of autofluorescence brought on by aldehyde fixation ⯈ TrueVIEW autofluorescence quenching kits with or with out DAPI distinction staining  |
| 5. Select mounting media with or with out distinction staining ⯈ VECTASHIELD Antifade mounting medium, with or with out distinction core staining  |
| 6. Visualization ⯈ Fluorescence microscope ⯈ View with appropriate excitation / emission filtersTonsils (FFPE): Cytokeratin detected by RTU VectaFluor Anti-Rabbit IgG, DyLight 488 package (inexperienced). Autofluorescence quenched with TrueVIEW Quenching and mounted with VECTASHIELD Vibrance Antifade Mounting Medium. |