TITLE: Immunofluorescence on Formalin-Fixed Paraffin-Embedded Tissue Sections
AUTHORS: Katarzyna  Dobaczewska, Zbigniew  Mikulski

[DESCRIPTION]
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.

[STEPS]
SECTION: Deparaffinization/ Rehydration
1. 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.
SECTION: Antigen Retrieval
2. 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.
SECTION: Freequenza Setup
3. 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. 





 

 

SECTION: Blocking
4.  Place 100µL of blocking buffer (5% normal serum (donkey) 0.3% triton X in PBS ) into coverplate slot and incubate for 1hr at RT Room temperature 



SECTION: Incubate with Primary Antibody
5. Place 100µL of optimal dilution of your purified antibody in PBS-T incubate at 4 °C overnight.
SECTION: Incubate with Secondary Antibody
6. 
 

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
SECTION: Counterstaining
7. 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
SECTION: Coverslipping
9. 
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.
SECTION: Autofluorescence Quenching
8.     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.