Mouse monoclonal igg 2 f11

Mice peripheral tissues and specific brain regions were fixed by vascular perfusion with 30 mL of 4 % paraformaldehyde (PFA) after perfusion with PBS for about 20 min. Desired organs were isolated and washed with PBS then processed using a rapid microwave automatic histo-processor using a standard protocol and embedded into paraffin wax. Subsequent immunohistochemistry assays were then performed per standard protocols previously described [15 (link)]. Briefly, sectioned tissues were deparrafinized then permeabilized with methanol. The sections were then blocked with 3 % BSA for 1 h at room temperature and later incubated with primary antibody (1:300 mouse monoclonal IgG 2 F11; Enzo Life Sciences Int., PA, USA and AntiCT)) at 4 °C overnight, washed with PBST then with secondary antibody conjugated to AlexFluor (1:600) for 2 h at room temperature. Counterstaining with Hoechst 33342 (1:10,000) was effected. The slides were then mounted by pristine mount and left to dry overnight at 4 °C. Images were collected by BZ-8000 microscope (KEYENCE, Osaka, Japan) with a × 20 Plan APO lens (Nikon, Tokyo, Japan).

Primary cortical cells on an eight-well Lab Tek chamber were fixed in 4 % PFA in PBS at 4 °C overnight followed by 3 wash steps with PBS. Fixed cells were permeabilized with 50 and 100 % methanol for 10 min each then washed with PBS 3 times. The permeabilized cells were washed with 0.1 % Triton X-100 in PBS (PBST), incubated in blocking buffer (3 % BSA in PBST) for 3 h at 4 °C, and then incubated in primary antibody (1 : 300; mouse monoclonal IgG 2 F11; Enzo Life Sciences Int., PA, USA and AntiCT) overnight at 4 °C. The cells were then washed with PBST and thereafter incubated in a secondary antibody conjugated to AlexFluor. The nuclei were visualized with Hoechst 33342. Images were collected using a BZ-8000 microscope (KEYENCE, Osaka, Japan) with a × 20 Plan APO lens (Nikon, Tokyo, Japan).