Immunofluorescence selection guide

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
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)
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
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.

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