TITLE: IMMUNOFLUORESENCE ANTIBODY (IF) STAINING PROTOCOL
AUTHORS: Scott Vermilyea

[DESCRIPTION]
This is the basic protocol for antibody staining of formalin fixed paraffin embedded (FFPE) tissue.

[GUIDELINES]
Principle:
For antibody staining to be successful, most FFPE tissue requires antigen retrieval of some kind.  Formalin fixation cross-links proteins during the course of fixation.  Antigen retrieval unlinks the proteins and opens up the antigen sites so that the antibody will be able to bind to them.

[STEPS]
SECTION: DAY 1: Deparaffinize tissue
1. Either place slides on a slide warmer with temperature set to approx. 57 °C.  Leave the slides on the warming plate until the paraffin looks melted on all of the slides (about 0-15 min).

SECTION: DAY 1: Deparaffinize tissue
2. Put slides in a 60 °C oven for about 30 min.  

 

 
 
 
SECTION: DAY 1: Preferred Antigen retrieval steps
3. Fill the vegetable steamer with deionized water to the second line in the transparent corner section of the steamer. 
SECTION: DAY 1: Preferred Antigen retrieval steps
4. Add capillary gap slides (same number as the number of slides you are staining) in the slide holder to wet the slides, or if you have a lot of slides, rinse the capillary gap slides separately before use. 
SECTION: DAY 1: Preferred Antigen retrieval steps
4.1. Fill the plastic “boat” containers with approx. 20 mL of one of the 1X antigen retrieval solutions (Reveal or citrate buffer).  Pick up a slide (rough side of the capillary gap slide facing the tissue section slide) and put the slides into one of the troughs in the boat container.  

SECTION: DAY 1: Preferred Antigen retrieval steps
4.2. Push slides toward each other and allow the fluid to come up slowly between the two slides.  Try not to get bubbles between the slides or this will obstruct the antigen retrieval process and give you uneven staining. 
SECTION: DAY 1: Preferred Antigen retrieval steps
4.3. Put the “boats” into the steamer and set for 35 min.  This will allow the steamer to come up to temperature and the slides will steam for about 30 min.

SECTION: DAY 1: Preferred Antigen retrieval steps
5. Remove clear basket from the steamer and allow slides to cool for about 20 min to room   temperature.

SECTION: DAY 1: Preferred Antigen retrieval steps
6. Transfer the “boats” of slides to the inner steamer container and rinse with running water for about 10-15 min.  Unpeel the slides from each other and transfer the slides to the gray slide holder for a final rinse (~5 min).

 


SECTION: DAY 1: Staining steps
7. Dilute or use prediluted TRIS (1x concentration )  for 10 min, approx 250 µL-300 µL per slide.

SECTION: DAY 1: Staining steps
8. 3% Hydrogen peroxide made in 1X TRIS for 10-20 min.

SECTION: DAY 1: Staining steps
9. Rinse slides in 1X TRIS and add to slides for 5 min.

SECTION: DAY 1: Staining steps
10. 100% Background Sniper for nearly 13 min.  Do not go longer than 15 min. (alternative: 10% normal goat serum made in 1X TRIS or PBS can be substituted for 60 min) or 1% sodium borohydride made in 1X TRIS soln to decrease autofluoresence.

SECTION: DAY 1: Staining steps
11. Make up antibody solution or a cocktail of multiple antibodies at desired concentration in a diluent of  5% Sniper in 1X TRIS solution.  Using approx. 100 µL per slide, make up enough antibody to cover all slides.

SECTION: DAY 1: Staining steps
12. Cover the slides with either a glass coverslip (24 x 60 mm) or parafilm and put in a 4 °Crefrigerator  .

SECTION: DAY 2:
13. Take slides out of the refrigerator and warm up to room temperature about 15-20 min.  Remove the cover slips and cover the slides with 1X TRIS solution for a few minutes.  Rinse off the antibody solution with 1X TRIS 5 min x 2.  At this stage you will need to use the black staining box to keep the reagents from light exposure.  

SECTION: DAY 2:
14. Secondary reagent/antibody for 1-120 min.
Apply the secondary reagent appropriate to the antibody, i.e., if your antibody was raised in a rabbit, you will apply a fluorescent antibody such as Jackson goat anti-rabbit 488 (yellow in tube, excites to green in IF light) using about250 µL-300 µL per slide or enough of the reagent to totally cover the tissue. 

SECTION: DAY 2:
14.1. If your antibody was raised in some other animal, you will have to find a secondary to that animal, i.e., donkey anti-mouse, goat anti-rat, etc.  Usually a dilution of  1:300 to 1:500 in a diluent of 5% Sniper made in 1X TRIS is a good starting concentration to use.

 


SECTION: DAY 2:
15. Rinse slides with 1X TRIS; 5 min x 3.

SECTION: DAY 2:
16. If you do not want to use the solutions to block autofluorescence, mount slides in dim light conditions with Prolong gold with DAPI.  The dapi will stain the nuclear elements blue.  Cover slides in a slide box or tin foil to dry overnight at room temperature to set the mounting media.
SECTION: DAY 2:
17. Blocking Autofluorescence (Optional step) with Vector TrueView.
SECTION: DAY 2:
17.1. In IHC 4 °Cfrig, TrueView kit on top shelf.

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17.2. Add equal volumes of reagents in order, mix A with B and mix together ~10 s.

SECTION: DAY 2:
17.3. Add C and mix again ~10 s.  Calculate approx. 100/slide.

SECTION: DAY 2:
17.4. Apply to tissue and incubate for 2-5 min.

SECTION: DAY 2:
17.5. Tissue will stain dark blue. Rinse with 1X Tris.
SECTION: DAY 2:
17.6. Stain with DAPI separately as in the following steps:


SECTION: DAY 2:
17.7. DAPI aliquots are in the -20 °C freezer and should be diluted as follow:

2.1 µL concentrated DAPI in 100 µL1X Tris.
Dilute the above solution 1:1000 in 1X Tris for the working solution of 1X DAPI.
Stain with the working solution of DAPI for 10 min.
Rinse in 1X TRIS a few times and coverslip with VectaShield Vibrance.
After coverslipping, cover slides and allow media to harden in the dark (Room temperature).



SECTION: DAY 2:
15.1. Rinse slides with 1X TRIS for 5 min (1/3).

SECTION: DAY 2:
15.2. Rinse slides with 1X TRIS for 5 min (2/3).
SECTION: DAY 2:
15.3. Rinse slides with 1X TRIS for 5 min (3/3).