Alexa fluor 594 affinipure donkey anti guinea pig igg

Mice were perfused with PBS and 4% PFA after behavioral testing. 30 μm mPFC sections were collected using a cryostat. Sections were incubated in blocking buffer (4% BSA + 0.3% Triton X-100 in PBS) for 1 hour at room temperature then transferred into primary antibody buffer (Anti-ProDynorphin antibody, ab10280, Abcam; 1:200 dilution in blocking buffer). Sections were incubated in primary antibody for 48 hours at 4 °C, then rinsed in PBS and transferred to secondary antibody (Alexa Fluor 594 AffiniPure Donkey Anti-Guinea Pig IgG, 706-585-148, Jackson ImmunoResearch; 1:200 dilution in blocking butter) for 2 hours at room temperature. Sections were then rinsed in PBS and mounted on slides. Fluorescence images were taken on Zeiss 780 confocal microscope with 20X objective. Data were quantified using Fiji (ImageJ) by analyzing fluorescence intensity in fixed-size regions of interest with intense GFP expression.

Sections were blocked overnight at 4°C in a PBS solution containing 2.0% TritonX-100 and 10% normal donkey serum and then incubated with primary antibody in a PBS-T solution (2% TritonX-100 and 10% normal donkey serum), overnight (24 h at 4°C). The next day, sections were rinsed in PBS-T and incubated in secondary antibody for 2 hr at room temperature in PBS-T solution (2% TritonX-100 and 10% normal donkey serum). Sections were mounted in Fluoromount-G (Southern Biotech). Primary Antibodies: Goat anti ChAT (Millipore, Cat# AB114P, 1:500), Rabbit Anti VAChT (Synaptic Systems, Cat# 139103, 1:500), Guinea-Pig Anti-cFos (Synaptic Systems, Cat# 226004, 1:500), Mouse-AT8 (Thermo Fisher, Cat# MN1020, 1:250), GP Anti ABeta (Synaptic Systems, Cat# 218308 , 1:500). Secondary antibodies: Donkey anti Goat IgG, Alexa Fluor 488 (Thermo Fisher, Cat# A32814, 1:750), Donkey anti Rabbit IgG, Alexa Fluor 647 (Thermo Fisher, Cat# A32795 1:750), Donkey anti Mouse IgG, Alexa Fluor 594 (Thermo Fisher, Cat# A32744, 1:750), Alexa-Fluor 647 AffiniPure Donkey Anti-Guinea Pig IgG (Jackson ImmunoResearch, Cat# 106-605-003, 1:750), Alexa-Fluor 488 AffiniPure Donkey Anti-Guinea Pig IgG (Jackson ImmunoResearch, Cat# 106-545-003, 1:750), Alexa-Fluor 594 AffiniPure Donkey Anti-Guinea Pig IgG (Jackson ImmunoResearch, Cat# 106-585-003, 1:750).