Natalie ChenBrecha Lab

9/26/15: Staining for TH, GABA, VGAT1. Frozen sections are thawed for 10 minutes on a tissue-warming block or thawed on

the bench. 2. Label the slide as they dry (antibody + dilution)3. Wash sections with 0.1 M Phosphate Buffer for 30 minutes.4. Non-specific sites blocked with blocking solution

a. line a tray with kim wipesb. fill with Millipore H2) so the wipes are dampc. take slide out off the holder and tap off the PBd. Use a PAP pen and circle the tissue so liquid stays insidee. add 200 ul (just enough to cover all of the sections)f. leave for 1 hour

5. Primary antibody solution prepared during block. a. Tyrosine Hydroxylaes MAb 52801:100 b. Tyrosine Hydroxylase Mab52801:300c. Allison Sargoy GABA (1:100)d. VGAT

i. 1:100ii. 1:500

iii. 1:10006. Samples are centrifuged for 5 minutes at max speed on eppendorf centrifuge. 10Kxg7. Block is removed from slide with a kimwipe and 250 ul of primary antibody is

added to each slide.8. The tissue is incubated at 4C overnight.