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 |