Immunofluorescence (IF) / Immunocytochemistry (ICC) Protocol

Seed cells onto sterile coverslips, chamber slides, or imaging plates so they reach 50-80 percent confluence on staining day. For coverslips in a 24-well plate, use enough medium to fully cover the cells.

Remove medium and gently rinse once with PBS. Add 4 percent paraformaldehyde in PBS for 10-15 minutes at room temperature. For some cytoskeletal, membrane, or epitope-sensitive targets, compare with cold methanol fixation for 5-10 minutes at -20 degrees C.

Wash 3 times with PBS, 5 minutes each. Permeabilize with 0.1-0.3 percent Triton X-100 in PBS for 5-10 minutes at room temperature. For nuclear histone or chromatin targets, 0.2-0.5 percent Triton X-100 can be tested. For membrane targets, reduce or omit detergent.

Add blocking buffer, such as 1-5 percent BSA or 5 percent normal serum in PBS with 0.05-0.1 percent Tween-20. Incubate 30-60 minutes at room temperature. Use serum from the host species of the secondary antibody when practical.

Dilute primary antibody in blocking buffer. Start with the datasheet-recommended IF/ICC dilution or test 1:100, 1:250, and 1:500. Add enough volume to cover the sample, such as 100-200 uL per coverslip or 50-100 uL per chamber well. Incubate 1 hour at room temperature or overnight at 4 degrees C in a humidified chamber.

Wash 3 times with PBS or PBST, 5 minutes each, using gentle rocking. Do not allow cells to dry.

Dilute fluorophore-conjugated secondary antibody in blocking buffer, commonly 1:500-1:1000. Add enough volume to cover the cells and incubate 45-60 minutes at room temperature protected from light.

Wash 3 times. Add DAPI or another nuclear counterstain for 5 minutes if desired, then wash once. Mount coverslips with antifade mounting medium and cure according to the mountant instructions.

Acquire images using the same exposure, laser power, gain, and offset for samples that will be compared. Include no-primary and secondary-only controls to evaluate background.

Use 4-8 um tissue sections mounted on charged slides. Air dry frozen sections for 10-20 minutes. For FFPE sections, deparaffinize through xylene and graded ethanol, then rehydrate to water.

For frozen sections, fix with cold acetone for 10 minutes or 4 percent paraformaldehyde for 10-15 minutes depending on the target. For FFPE sections, perform heat-induced antigen retrieval in citrate buffer pH 6.0 or Tris-EDTA pH 9.0 for 10-20 minutes, then cool 20 minutes.

For tissue with high autofluorescence, treat with an autofluorescence quenching reagent or freshly prepared 0.1 percent sodium borohydride for 5-10 minutes, followed by thorough PBS washes. Choose the method compatible with your fluorophores.

Permeabilize with 0.1-0.3 percent Triton X-100 in PBS for 5-10 minutes if intracellular or nuclear access is needed. Block 30-60 minutes with 5 percent normal serum or 1-5 percent BSA in PBS.

Dilute primary antibody in blocking buffer. Start with the datasheet-recommended tissue IF dilution or test 1:100, 1:250, and 1:500. Apply 100-200 uL per section, cover with parafilm or use a humidified chamber, and incubate 1-2 hours at room temperature or overnight at 4 degrees C.

Wash 3 times for 5 minutes each in PBS or PBST. Add fluorophore-conjugated secondary antibody diluted 1:500-1:1000. Incubate 45-60 minutes at room temperature protected from light.

Wash 3 times. Add DAPI if desired. Mount with antifade medium and coverslip carefully to avoid bubbles.

Use identical microscope settings for comparative samples. Include no-primary controls and known positive tissue when available.

Choose primary antibodies from different host species when possible. Match secondary antibodies to non-overlapping fluorophores. Avoid fluorophores with heavy spectral overlap unless spectral unmixing is available.

Prepare the sample using the IF/ICC or tissue IF fixation and blocking conditions appropriate for the most sensitive target. Use enough blocking buffer to reduce nonspecific secondary antibody binding.

Prepare a master mix containing each primary antibody at its optimized dilution. Include primary antibody if it is one of the targets in the panel. Start with single-stain optimized dilutions before combining antibodies. Incubate 1 hour at room temperature or overnight at 4 degrees C.

Wash 3 times for 5 minutes each with PBS or PBST. Extend washes to 5-10 minutes when background or cross-channel signal is high.

Add cross-adsorbed secondary antibodies diluted in blocking buffer, commonly 1:500-1:1000. Incubate 45-60 minutes protected from light.

Wash 3 times. Add nuclear counterstain if needed, then mount with antifade medium compatible with all fluorophores.

Image single-color controls, no-primary controls, and full-stain samples using consistent settings. Use single-color controls to identify bleed-through or compensation needs.

Plan untreated, stimulated, inhibited, knockdown, or positive control samples before staining. For phospho targets, add phosphatase inhibitors during washes or lysis-related steps where applicable and fix quickly after treatment.

Remove medium and rinse briefly with warm PBS when needed. Fix with 4 percent paraformaldehyde for 10-15 minutes at room temperature, or use methanol fixation for targets known to work better with alcohol fixation.

Wash 3 times. Use 0.1-0.3 percent Triton X-100 for 5-10 minutes. For histone or chromatin PTMs, test 0.2-0.5 percent Triton X-100 and consider a short pre-extraction step only if compatible with morphology and the target.

Block 45-60 minutes with 1-5 percent BSA in PBS or TBS. For phospho-specific antibodies, BSA is often preferred over milk because milk can contain phosphoproteins that increase background.

Dilute primary antibody in blocking buffer. Start with the datasheet-recommended IF dilution or test 1:100, 1:250, and 1:500. Incubate overnight at 4 degrees C for low-abundance PTMs or weak signals.

Wash 3 times for 5 minutes. Add fluorophore-conjugated secondary antibody at 1:500-1:1000 for 45-60 minutes protected from light. Wash 3 times.

Counterstain if needed, mount with antifade medium, and image positive and negative controls with the same settings. For signal quantification, avoid saturated pixels and analyze the same cellular compartment across samples.