Feb 18, 2022
Immunofluorescence on Formalin-Fixed Paraffin-Embedded Tissue Sections
- 1La Jolla Institute for Immunology
- La Jolla Institute Microscopy and Histology Core
Protocol Citation: Katarzyna Dobaczewska, Zbigniew Mikulski 2022. Immunofluorescence on Formalin-Fixed Paraffin-Embedded Tissue Sections. protocols.io https://dx.doi.org/10.17504/protocols.io.b49yqz7w
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: February 17, 2022
Last Modified: June 06, 2024
Protocol Integer ID: 58392
Abstract
Fluorescence staining of FFPE tissues that leads to consistent and quality results.
Guidelines
Make sure all reagents used in the Freequenza rack are at room temperature to avoid air bubble formation that can affect the quality of your stain.
For antigen retrieval citrate gives better signal to noise ratio in most cases. However, some antibodies will only work with Tris-EDTA buffer.
When first optimizing a new antibody run the following controls.
a. Unstained slide ( Autofluorescence control)
b. Isotype control
c. Secondary only control
d. A couple of dilutions of your antibody.
Materials
0.1% Tween-20-containing 1XPBS solution
Prolong GoldThermo Fisher ScientificCatalog #P36930
Pro-Par ClearantAnatech LTPCatalog #510
Hoechst 33342Catalog #H3570
TrueBlack® Lipofuscin Autofluorescence QuencherBiotiumCatalog #23007
Materials:
Reagents:
-Pro-par Clearant
-Reagent alcohol
-Antigen Retrieval Buffer: Citrate (PH 6.0) (10mM Sodium Citrate .05% Tween 20 pH 6.0) or Tris-EDTA buffer( pH 9.0)(10mM Tris Base 1mM EDTA .05% Tween 20 9.0)
-Wash Buffer: 1X PBS-T ( .1% tween 20 detergent )( 1ul/mL)
-Blocking solution : 5% normal serum ( donkey ) 0.3% triton X in PBS
-Primary antibody of choice
-Secondary antibody of choice
-Hoechst 33342
-True Black Autofluorescence Quencher
-Prolong gold
Lab equipment:
- Epredia™ Shandon™ Plastic Coverplates Cat no.72-110-017
- Decloaking Chamber™ NxGen with slide racks and metal slide canister
- Freequenza
-Coplin Jar
Deparaffinization/ Rehydration
Deparaffinization/ Rehydration
a. Bake slides for 1 hour at 60 °C
b. Dip slides in Propar 20 times then let sit for 10 min. Repeat step 3x, each time with fresh Propar.
c. Dip slides in 100% reagent alcohol 20 times then let sit for 1 min 30 sec. Repeat step with fresh alcohol.
d. Dip slides in 90% reagent alcohol 20 times then let sit for 1 min 30 sec.
e. Place the slides under running DI water for 2 min.
Antigen Retrieval
Antigen Retrieval
a. Place slides in a rack into the Biocare Medical metal slide canister
b. Fill canister with Citrate antigen retrieval buffer.
b. Use the Biocare Medical decloaking chamber on program 5.
c. After program completion remove from chamber and let cool on the lab bench for 30 min.
d. Move rack into a fresh container, fill with PBS-T and let sit for 2 min.
Freequenza Setup
Freequenza Setup
b. Cover slides with plastic Shandon coverplates and place them into the Freequenza rack.
c. Wash with 1mL of PBS-T and watch the flowrate through the Freequenza apparatus. If any coverplate slot appears to drain faster than the rest remove it from Freequenza rack and reapply the coverplate. If issue persists dispose of coverplate.
Note
Place Slide on the feet at the bottom of the coverplate with the tissue facing towards the coverplate. Do this step under PBS ( First portion of the video is to demonstrate what to do in the PBS buffer).
Check for air bubbles and that the slide is properly placed. If air bubbles are present redo this step.
Snap into place on the freequenza rack.
Check the flow rate.
Proceed with staining.
Blocking
Blocking
Place 100µL of blocking buffer (5% normal serum (donkey) 0.3% triton X in PBS ) into coverplate slot and incubate for 1hr at Room temperature
Incubate with Primary Antibody
Incubate with Primary Antibody
Place 100µL of optimal dilution of your purified antibody in PBS-T incubate at 4 °C overnight.
Incubate with Secondary Antibody
Incubate with Secondary Antibody
Note
Before proceeding with secondary antibody incubation let Freequenza rack equilibrate to room temperature for an hour.
a. Wash 4x with 1mL of PBS-T
b. Incubate with 100µL secondary antibody diluted to optimal concentration in PBS-T for 1hr at Room temperature
c. Wash 4x with 1mL of PBS
Counterstaining
Counterstaining
a. Place 100uL of Hoechst 1:1000 (PBS) ( 10mg/mL stock) on each slide, incubate for 5 min.
b. Wash 3X with 1mL of PBS
Autofluorescence Quenching
Autofluorescence Quenching
a. Working in small batches remove slides from Freequenza and lay flat
b. Tap excess PBS off the slides
c. Overlay slides with TrueBlack Lipofuscin Autofluorescence Quencher (diluted 1:20 with 70% ethanol) incubate for approximately 30 seconds.
d. Place slides into a Coplin jar and fill with PBS. Let sit for 10 minutes.
e. Overfill the jar to allow residual TrueBlack to come to the top of the buffer surface. Discard the buffer from Coplin jar and refill it. Let sit for 10 min. Repeat step.
Coverslipping
Coverslipping
Coverslip with 2-3 drops of ProLong Gold antifade mounting medium without DAPI
Remove excess mounting media: Using a lab vacuum aspirator pull the excess from the edges of the cover glass.
Lay flat and allow to dry for at least 24 hours in the dark.