Jun 11, 2026
Version 3
Immuno-Fluorescence Staining of Paraffin sections - v3 V.3
- Domenic Filingeri1,2
- 1University of Pittsburgh;
- 2Children's Hospital of Pittsburgh
Protocol Citation: Domenic Filingeri 2026. Immuno-Fluorescence Staining of Paraffin sections - v3. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6ex9rgqe/v3Version created by Domenic DF Filingeri
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: March 02, 2026
Last Modified: June 11, 2026
Protocol Integer ID: 244261
Keywords: paraffin section, immunofluorescence, dapi step, fluorescence staining of paraffin section, fluorescence staining, immuno
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
Day 1
5h 47m
Select paraffin slides for IF staining.
Note
Make sure to have secondary-only negative controls.
60 °C for 00:25:00 in dry incubator
Use glass slide box
25m
Re-hydrate with HistoClear and Ethanol.
Create each as a 50mL conical tube, pouring into glass slide box for each step.
Pour slowly and not directly onto slides.
HistoClear 00:10:00 00:10:00 X 2 times
100% EtOH 00:05:00 00:05:00 X 2 times
90% EtOH 00:03:00 , 45 mL pure EtOH with 5 mL DI water
80% EtOH 00:03:00 40 mL pure EtOH in 10 mL DI water
70% EtOH 00:03:00 35 mL pure EtOH in 15 mL DI water
50% EtOH 00:03:00 , 25 mL pure EtOH in 25 mL DI water
42m
Wash in tap water, 00:05:00 00:05:00 00:05:00 X 3 times
15m
Antigen retrieval. Transfer slides to plastic slide box, now, fill to line with retrieval buffer.
Select one Antigen Retrieval buffer method from below. (full recipe below)
1 mM EDTA, pH 8.0
Tris-EDTA buffer, pH 9.0
Sodium Citrate, pH 6.0
Required
Antigen Retrieval ReagentAdd DI-water to pressure cooker up to metal plate. (Slides in plastic box with retrieval buffer)
Presser cooker on porridge option, which is 00:20:00
Remove after 00:45:00 . Display will read "LO -25" --> which means 25m after porridge completes
45m
Move slides to sit at Room temperature (in plastic box + retrieval buffer)
Allow to cool to Room temperature slowly
This can take up to 02:00:00 . (Minimum 00:30:00 ), until box warm/cool to touch.
2h
Rinse with tap water, 00:05:00 00:05:00 X 2 times
10m
Wipe away water, circle section with slide pen, then re-add 200uL/slide of tap water.
Transfer slides to black hydration box.
Note
Cut P-200 pipette tip to prevent high pressure for subsequent steps
Use 200uL for all future steps unless noted, aspirating with vacuum
Permeabilize nuclear membrane with 0.25% Triton X-100 in PBS 00:05:00 to 00:10:00
10m
Wash with PBST/ TBST 00:10:00 00:10:00 X 2 times
20m
Incubate slides with blocking buffer in hydration chamber for
00:30:00 - 01:00:00 at Room temperature
Note
Amit's: blocking buffer is 5%BSA in TBST
Usually primary antibody is at [1:400]
1h
Rinse in PBST/ TBST briefly
Incubate for primary antibody diluted in blocking buffer
Leave Overnight at 4 °C in hydration chamber.
Add tap water to wells between slides in chamber to prevent evaporation
Required
Species-AntigenRequired
ConcentrationCompany/ Product #
Note
Amit: Antibody diluted in 3%BSA in TBST
Day2
1h 57m
Double wash in PBST/ TBST 00:03:00 00:03:00 00:03:00 X 3 times.
9m
Incubate with secondary antibody diluted in 1x PBS/ blocking buffer at Room temperature for 01:00:00 in dark (min 30') in hydration chamber.
Turn lab lights off to apply secondary.
Required
Species + FluorophoreRequired
ConcentrationCompany/ Product #
Note
Amit's Antibody diluted in 3%BSA in TBST
Usually secondary is [1:500], with GFP/488 at [1:1000]
1h
Double wash in PBST/TBST 00:03:00 00:03:00 00:03:00 X 3 times.
9m
Incubate with 1x DAPI at Room temperature for00:15:00 in a hydration chamber.
Dilute 1:1000 in ddH2O
Note
DAPI located in Dom's -4*C box at 1000X
15m
Double wash in PBST/ TBST 00:03:00 00:03:00 00:03:00 X 3 times.
9m
Add one small drop anti-fade mounting medium 10-15 µL to each section, cover, press with plastic pipette tip to remove bubbles, blot clean with paper towel. Store dark at Room temperature in dark at least 00:15:00 , then move to 4 °C DRY slide box for Overnight .
15m
Trace edge of slide cover with clear nail polish, then return to 4 °C DRY slide box
Note
Nail polish adds to longevity of slides by preventing dry-out.
Reagents
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.
Sodium Citrate, pH 6.0
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
Blocking buffer
10% Normal Serum
1% BSA
0.25% NP40
In 1x PBS
PBST
990 mL PBS
0.1% Tween 20 -- 10 mL (10% Tween 20) or 1mL (100% tween)
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
0.25%Triton X 100 in PBS
10x PBS (900 mL Pure, 100 mL PBS 10X )
2.5 mLTriton X 100
25 mL 10x Triton
TBST
50ml of 20x TBS
950ml DI H2O
1ml tween (cut tip to ensure you get one full ml, add to bottle)
Acknowledgements
Edited by Jeong Lee on 6/28/2022
Edited by Domenic Filingeri/ uploaded to Protocols.io Jan 2026