Immunohistochemistry
Peter Kilfeather
Abstract
This protocol describes the immunohistochemistry protocol.
Steps
Brain Preparation
Euthanize the mouse by lethal intraperitoneal injection of pentobarbital.
Upon loss of the pedal withdrawal reflex, dissect the thoracic cavity open.
Perfuse 25 mL phosphate-buffered saline (PBS) transcardially, followed by 25 mL 4 % paraformaldehyde (PFA), diluted in PBS.
Tissue Collection and Storage
Dissect and extract the brain.
Store the brain in 4 % PFA at 4 °C overnight.
Dehydration
Slice each brain into 3 mm coronal sections (approximately).
Pass each section through graded alcohol solutions (50, 70, 80, 95, 100, 100 and 100 % ethanol), holding for 10 minutes in each.
Transfer each section into xylene three times, for 20 minutes each.
Transfer each section into paraffin two times, for two hours each.
Embed each section in fresh paraffin, orienting as desired.
Sectioning
Take coronal sections at 7 µm using a Leica microtome (Leica Microsystems, Wetzlar, Germany) and mounted on glass slides (VWR, Superfrost Plus)
Deparaffinisation, Antigen Retrieval and Blocking
Transfer each slide-mounted set of sections through xylene (twice) and reversed graded alcohols (100, 95, 70, 50 %) for 10 minutes each.
Transfer to 1X citrate buffer (ab96678, Abcam) and perform antigen retrieval by microwave heat (20 minutes).
Block for one hour at room temperature using 10 % donkey serum in tris-buffered saline (TBS).
Primary Staining
Apply primary antibodies to TH (Abcam Cat# ab76442, RRID:AB_1524535, 1:500), GFP (Thermo Fisher Scientific Cat# A-11122, RRID:AB_221569, Invitrogen, 1:1000), CASR (Abcam Cat# ab79038, RRID:AB_2071489, 1:100) or TYROBP/DAP12 (Abcam Cat# ab283679, 1:100), prepared in 1 % donkey serum in TBS and incubate overnight at 4 °C
Secondary Staining
Wash slides in TBS three times, for five minutes each.
Incubate in secondary antibodies (Alexa Fluor-conjugated anti-rabbit/chicken A-31573, RRID:AB_2536183 and A78952, RRID:AB_2921074 1:1000) and DAPI (Thermo Fisher Scientific Cat# D3571, RRID:AB_2307445, 1:5000), prepared in TBS for 1 hour at room temperature.
Wash slides in TBS three times, for five minutes each.
Mount coverslips using FluorSave mounting medium (Millipore, Massachusetts, United States of America).
