Tissue preparation and storage

We use this protocol on tissue from a perfused animal (mouse or goldfish, usually). Tissue is postfixed in 4% PFA for 2-4 hours, then placed in 20% sucrose in 0.1M PB overnight at 4C.

Tissue is cut on cryostat between 12 and 18 microns and collected on superfrost plus slides (Fisher). Tissue slides are then stored at –20C. Let slides sit in freezer for at least one night before processing. (We put slides in slide box and wrap the box in a plastic bag or parafilm.)

Immunohistochemistry

DAY 1

Remove slides from –20 and let come to room temperature (5-10minutes...I usually label my slides with animal # etc. after I remove them from the freezer and by the time I choose my slides and label them, they are at room temp)

Place slides in 0.1M PB

Dry on hot plate at ~40C (not everyone does this step, but I find it helps the sections stay on the slides). Remove slides from PB, dry the back of the slide with a Kimwipe, and place slide on hot plate for a few minutes until tissue is almost dry but not crispy.

Return to the same PB until all slides are dry.

Wash 3 x 10 minutes in 0.1M PBS

1-2 hour block in blocking solution (see next page) and 1-3% normal serum-calculate 200ul per slide (use the serum from the animal your secondary antibody is made in). Coverslip slides with parafilm and let sit at room temp.

When blocking is almost done, prepare the primary antibody solution. Calculate enough for 200ul per slide. Dilute primary antibody in blocking solution with the same normal serum used above. Below is a list of some commonly used primary antibodies and their dilutions.

5HT Biomeda/mouse 1:1 (see 5HT protocol for more info)

5HT Biomeda/rabbit 1:1

Bgal Cindy Yee/guinea pig 1:1000

CGRP Pennisula/rabbit 1:1000

GLAST Chemicon/guinea pig 1:500 (use antigen retrieval + TSA)

Gustducin Santa Cruz/ rabbit 1:1000

NGF Santa Cruz/rabbit 1:500

NTPDase1 Sevigny/rabbit 1:1000

NTPDase2 Sevigny/rabbit 1:1000

NTPDase3 Sevigny/guinea pig 1:1000

P2X2 Alomone/rabbit 1:500

P2X3 Chemicon/rabbit 1:500

PGP Biogenesis/rabbit 1:1000

PLCB2 Santa Cruz/rabbit 1:1000

SNAP 25 Calbiochem/rabbit 1:200

T1R3 Margolskee/rabbit 1:500

Remove coverslips, drain blocking solution from slides, and apply primary antibody solution and coverslip with parafilm. Let incubate overnight at 4C.

DAY 2

Remove coverslips, drain slides and wash 3 x 10 min in PBS

Dilute secondary in blocking solution (we usually use Alexa secondaries from Molecular Probes and dilute them 1:400)

Cover slides with secondary solution, coverslip with parafilm, let incubate 2 hours at room temp.

Remove coverslips, drain solution and wash 1 x 10 minutes PBS

The last two ten-minute washes use PB

Mount with Fluormount G

Blocking Solution Stock

to make 100mL

50mL 0.2M PB

3mL 5 M NaCl

3mL 10% triton

44mL dH2O

100mg Bovine Serum Albumin (BSA)

We make up a liter at a time, aliquot in 50mL tubes, and freeze at –20. We defrost aliquots as we need them and store at 4C. Add normal serum only to what is needed for an experiment.