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
⯈ 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
⯈ One-mark. Fast. Conveniently.
⯈ Fluorophore-conjugated secondary antibodies
⯈ VectaFluor RTU DyLight labeled secondary antibodies
Dual-labeling, detection of two antigens.
⯈ VectaFluor Duet IF kits for double marking
⯈ Based on biotin.
⯈ Biotinylated secondary antibody and fluorophore conjugated to avidin or streptavidin.
⯈ See step Three for extra amplification
⯈ 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)
Více 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
⯈ 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.