Immunofluorescence (Protein Modality)¶

Multi-modal data

Immunofluorescence (IF) provides protein-level spatial information, complementing the RNA-level data from PRISM/SPRINTseq. This enables multi-modal analysis of the same tissue section (RNA + protein).

Applicable samples: Fresh Frozen cryosections (post-RNA detection or standalone)
Version: v1.0
Last modified: 2026-04-03

Post-RNA detection IF

This protocol is designed for samples that have completed RNA detection (e.g., PRISM / SPRINTseq) and require subsequent protein immunofluorescence imaging. It can also be used as a standalone IF protocol for cryosections.

1. Fixation¶

Goal: Cross-link proteins to preserve antigenicity and tissue morphology.

Fixative: 4% PFA at room temperature (RT) for 30 min.

2. Wash¶

Goal: Remove residual fixative.

Wash: PBST (PBS + 0.1% Tween-20), 5 min × 3 washes.

3. Blocking¶

Goal: Block nonspecific antibody binding.

Blocking solution: Beyotime Immunostaining Blocking Buffer (碧云天免疫荧光封闭液).
Incubate: RT for 2 h.

4. Primary Antibody Incubation¶

Goal: Specific antigen binding.

Dilution: 1:200 in Solarbio Quick Antibody Dilution Buffer.
Incubate: 4°C overnight.

Do not let the section dry

Ensure the section remains covered with solution throughout incubation. Use a humidified chamber.

5. Primary Antibody Wash¶

Goal: Remove unbound primary antibody.

Aspirate primary antibody solution as thoroughly as possible.
Wash: PBS, 15 min × 3 washes.

6. Secondary Antibody Incubation¶

Goal: Fluorescent labeling of bound primary antibody.

Secondary antibody: IgG-Alexa Fluor 488 conjugate.
Dilution: 1:200 in Solarbio Quick Antibody Dilution Buffer.
Incubate: RT for 2 h, protected from light.

Light protection

From this step onward, keep samples protected from light to prevent photobleaching of the fluorophore.

7. Secondary Antibody Wash¶

Goal: Remove unbound secondary antibody.

Wash: PBS, 15 min × 1 wash.

Quick Reference¶

Step	Reagent	Condition	Duration
Fixation	4% PFA	RT	30 min
Wash	PBST	RT	5 min × 3
Blocking	Beyotime Blocking Buffer	RT	2 h
Primary Ab	1:200 in Solarbio Quick Dilution Buffer	4°C	Overnight
Wash	PBS	RT	15 min × 3
Secondary Ab	IgG-488, 1:200 in Solarbio Quick Dilution Buffer	RT, dark	2 h
Wash	PBS	RT	15 min × 1