Table 1.

List of typical experimental variables affecting the outcome of an immunofluorescent reaction

1. Varying the duration of perfusion fixation from 10 to ≥25 min
2. Altering the fixative from aldehyde to methanol, acrolein or acetone
3. Varying the concentration of paraformaldehyde (from 1 to ≥2%) and glutaraldehyde (from 0 to ≥0.05%)
4. Changing the type of buffer in which the aldehyde is diluted from phosphate to borate, acetate, citrate or cacodylate and a corresponding change in pH from 6.0 to 8.0
5. Varying the duration of postfixation from 0 to ≥30 min
6. Applying antigen retrieval methods (e.g., microwave irradiation, heat treatment or enzymatic digestion)
7. Using free-floating vibratome sections versus cryostat sections attached to glass histological slides
8. Using freeze-thawing or not
9. The presence or absence of detergents in the blocking, primary and secondary Ab solutions
10. Using different molecules for blocking nonspecific labeling (e.g., normal serum, fish skin gelatin, bovine serum albumin, fetal calf serum, milk powder, etc.)
11. Changing the primary Ab dilution from 1:50 to ≥1:500
12. Changing the incubation time from overnight at room temperature to ≥2 d at 4°C
13. Changing the secondary Ab dilution from 1:50 to ≥1:500