IMMUNOFLUORESENCE ANTIBODY (IF) STAINING PROTOCOL

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 0-0h 15m 0s).

Put slides in a 60°C oven for about 0h 30m 0s.

A	B
Xylene	5 minutes
Xylene	5 minutes
Xylene	10 minutes
100% Alcohol	3 - 5 minutes
100% Alcohol	3 - 5 minutes
95% Alcohol x 2	3 - 5  minutes
80% Alcohol	3 - 5 minutes
Filtered water	3 - 5 minutes

Fill the vegetable steamer with deionized water to the second line in the transparent corner section of the steamer.

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.

Fill the plastic “boat” containers with approx. 20mL 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.

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.

Put the “boats” into the steamer and set for 0h 35m 0s. This will allow the steamer to come up to temperature and the slides will steam for about 0h 30m 0s.

Remove clear basket from the steamer and allow slides to cool for about 0h 20m 0s to room temperature.

Transfer the “boats” of slides to the inner steamer container and rinse with running water for about 10-0h 15m 0s. Unpeel the slides from each other and transfer the slides to the gray slide holder for a final rinse (~0h 5m 0s).

Dilute or use prediluted TRIS (1x concentration ) for 0h 10m 0s, approx 250µL-300µL per slide.

3% Hydrogen peroxide made in 1X TRIS for 10-0h 20m 0s.

Rinse slides in 1X TRIS and add to slides for 0h 5m 0s.

100% Background Sniper for nearly 0h 13m 0s. Do not go longer than 0h 15m 0s. (alternative: 10% normal goat serum made in 1X TRIS or PBS can be substituted for 1h 0m 0s) or 1% sodium borohydride made in 1X TRIS soln to decrease autofluoresence.

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. 100µL per slide, make up enough antibody to cover all slides.

Cover the slides with either a glass coverslip (24 x 60 mm) or parafilm and put in a 4°Crefrigerator .

Take slides out of the refrigerator and warm up to room temperature about 15-0h 20m 0s. Remove the cover slips and cover the slides with 1X TRIS solution for a few minutes. Rinse off the antibody solution with 1X TRIS 0h 5m 0s x 2. At this stage you will need to use the black staining box to keep the reagents from light exposure.

Secondary reagent/antibody for 1-2h 0m 0s.

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 about250µL-300µL per slide or enough of the reagent to totally cover the tissue.

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.

Rinse slides with 1X TRIS; 5 min x 3.

Rinse slides with 1X TRIS for 0h 5m 0s (1/3).

Rinse slides with 1X TRIS for 0h 5m 0s (2/3).

Rinse slides with 1X TRIS for 0h 5m 0s (3/3).

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.

Blocking Autofluorescence (Optional step) with Vector TrueView.

In IHC 4°Cfrig, TrueView kit on top shelf.

Add equal volumes of reagents in order, mix A with B and mix together ~0h 0m 10s.

Add C and mix again ~0h 0m 10s. Calculate approx. 100/slide.

Apply to tissue and incubate for 2-0h 5m 0s.

Tissue will stain dark blue. Rinse with 1X Tris.

Stain with DAPI separately as in the following steps:

DAPI aliquots are in the -20°C freezer and should be diluted as follow:

• 2.1µL concentrated DAPI in 100µL1X 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 0h 10m 0s.
• 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).