Jan 16, 2026
Version 1
Immunofluorescence staining –Paraffin sections V.1
- Domenic Filingeri1
- 1University of Pittsburgh
Protocol Citation: Domenic Filingeri 2026. Immunofluorescence staining –Paraffin sections. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6ex9rgqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 14, 2026
Last Modified: January 16, 2026
Protocol Integer ID: 238596
Keywords: paraffin section, immunofluorescence, dapi step
Abstract
IF staining for paraffin sections (DAPI step included)
Materials
- HistoClear
- Ethanol (100%, 95%, 90%, 80%, 70%, 50%)
- Tap water
- Triton X-100
- PBS
- Sodium citrate buffer (10 mM Sodium citrate, 0.05% Tween 20, pH 6.0)
- EDTA buffer pH 8.0
- Tris-EDTA buffer pH 9.0
- Blocking buffer
- Primary antibody
- Secondary antibody
- 1xDAPI
- Anti-fade mounting medium
- Sodium citrate (dihydrate) 2.94 g
- Distilled water
- 1N HCl
- Tween 20
- EDTA 0.37 g
- NaOH
- Tris 1.21 g
- BSA
- NP40
- Normal Serum
Troubleshooting
60°C for 25 min in dry incubator
25m
Re-hydrate with HistoClear and Ethanol, create each as a 50mL conical tube, pour into glass slide box. Pour slowly and not directly onto slides.
HistoClear 10 min X 2 times
100% EtOH 5 min X 2 times
90% EtOH 3 min, (45mL in 5mL DI water)
80% EtOH 3 min, (40mL in 10mL DI water)
70% EtOH 3 min, (35mL in 15mL DI water)
50% EtOH 3 min, (25mL in 25mL DI water)
45m
Wash in tap water, 5 min X 3 times
15m
Antigen retrieval. Select one method from below. Use plastic slide box
Sodium citrate (pH 2.0, pH6.0, or pH 9.0)
Tris-EDTA buffer pH 9.0
EDTA buffer pH 8.0
Heat them using a presser cooker for 20 minutes in plastic box. Add DI-water to pressure cooker up to metal plate. Select porridge option, remove at "LO 25min", then RT for 1 hour
45m
Allow to cool to RT slowly. This can take up to 2 hours. (Minimum 30 min)
2h
Wash in tap water, 5 min X 2 times
10m
Wipe away water, circle section with pap-pen, then add 200uL/slide of tap water. (Use 200uL for all future steps unless noted)
cut P-200 pipette tip to prevent high pressure for subsequent steps
Permeabilize nuclear membrane with 0.25% Triton X-100 in PBS 5 min
5m
Wash in tap water 10 min X 2 times
20m
Incubate slides with blocking buffer for 30min - 1 hour at RT in hydration chamber (add tap water to wells between slides).
1h
Blocking buffer is 5%BSA in TBST
Rinse in PBST briefly
Incubate for primary antibody diluted in blocking buffer. Leave overnight at 4°C in hydration chamber. (add tap water to wells between slides).
Antibody diluted in 3%BSA in TBST
Wash in PBST 3 min X 3 times. Double wash
10m
Incubate with secondary antibody diluted in 1x PBS at RT for 30 min to one hour in hydration chamber (dilute in PBS).
1h
Antibody diluted in 3%BSA in TBST
Wash in PBST 3 min X 3 times. Double wash
10m
Incubate with 1xDAPI at RT for 15 min in a hydration chamber. (Dilute 1:100 in ddH2O)
15m
Wash in PBST 3 min X 3 times. Double wash
10m
Add one small drop anti-fade mounting medium (10-15uL) to each section, cover, press with plastic pipette tip to remove bubbles, blot clean with paper towel. Store at least 15', then move to 4*C
1 mM EDTA, pH 8.0
EDTA 0.37 g
Distilled water 1 L
Adjust to pH 8.0 with NaOH
Store at room temperature for 3 months
Tris-EDTA buffer (10 mM Tris base, 1 mM EDTA solution, 0.05% Tween 20, pH 9.0)
Tris 1.21 g
EDTA 0.37 g
Distilled water 1 L
Mix to dissolve. Adjust pH to 9.0.
Add 0.5 mL of Tween 20 and mix well. Store at room temperature for 3 months or at 4°C for longer storage.
Blocking buffer
10% Normal Serum
1% BSA
0.25% NP40
In 1x PBS
PBST
1x PBS
0.1% Tween 20
4x Blocking Buffer stock (50 ml)
0.5g BSA in 40 ml of 1xPBS. Mix to dissolve. It can be warmed in a water bath.
Add 1.25 ml of 10% NP40 and mix by inverting. Do not vertexing. It causes a lot of bubbles.
Add 1x PBS up to 50 ml Vol.
Aliquot (250 ul in 1.5 ml tube) and store at –20 C.
Working blocking buffer
250ul of 4x blocking buffer
100 ul of normal serum (if all secondary antibodies are from one species)
650 ul of 1x PBS
Sodium Citrate
2.94g Tri-Sodium Citrate
800 mL distilled (pure) Water
Mix, dissolve
Adjust pH to 6.0 with 1M HCI
Top off to 200 mL using Pure diH2O
Add 0.5 mL Tween 20, or 5mL 10% Tween
PBST
990 mL PBS
10 mL (10% Tween 20) or 1mL (100% tween)
.25%Triton X 100 in PBS
10x PBS (900 mL Pure, 100 mL PBS 10X )
2.5 mLTriton X 100
25 mL 10x Triton
Acknowledgements
Edited by Jeong Lee on 6/28/2022