Mouse monoclonal anti iba1 antibody

Immunohistochemical staining of 7 µm sections of full-thickness retinas was performed by immunofluorescence with the following primary antibodies: mouse monoclonal anti-GFAP antibody (1:200; Cell Signaling Technology, Inc., Danvers, MA, USA) and mouse monoclonal anti-Iba1 antibody (1:150; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Seven days after ONC, the eyes were enucleated and fixed in freshly made 4% paraformaldehyde in 0.1 M PBS for 1 h, and then the free retina was harvested and flattened. The tissues were dehydrated and embedded in paraffin, and the retinal cross sections were then obtained. The tissues were blocked with 1% bovine serum albumin in PBS for 1 h at room temperature to prevent nonspecific background staining, and then with the primary antibodies overnight at 4 °C. The sections were washed several times with PBS, incubated with Alexa Fluor^® 488-conjugated chicken anti-mouse IgG (1:250; Invitrogen, Carlsbad, CA, USA) or Alexa Fluor^® 546-conjugated rabbit anti-goat IgG (1:250; Invitrogen, Carlsbad, CA, USA) for 4 h at 4 °C, and then washed again with PBS. The sections were counterstained with 0.1 µg/mL of Hoechst 33,342 stain in PBS (Invitrogen, Carlsbad, CA, USA). The images were analyzed using a Zeiss LSM 800 confocal microscope (Carl Zeiss, Jena, Germany).

Immunohistochemical staining of 7-µm wax sections of full-thickness retinas was performed by immunofluorescence with the following primary antibodies: mouse monoclonal anti-GFAP antibody (1:200; Cell Signaling Technology), mouse monoclonal anti-Iba1 antibody (1:150; Santa Cruz Biotechnology), mouse monoclonal anti-HIF-1α (1:200; Cell Signaling Technology), and rabbit monoclonal anti-acetyl-H3 (1:200; Cell Signaling Technology). Tissues were blocked with 1% bovine serum albumin in PBS for 1 h at room temperature to prevent nonspecific background staining, and then with primary antibodies overnight at 4 °C. The sections were washed several times, incubated with Alexa Fluor 488–conjugated chicken anti-mouse IgG (1:250; Invitrogen, Carlsbad California) or Alexa Fluor 546-conjugated rabbit anti-goat IgG (1:250; Invitrogen) for 4 h at 4 °C, and then washed again with PBS. The sections were counterstained with Hoechst 33342/PBS (0.1 µg/mL; Invitrogen). Images were analyzed using a Zeiss LSM 510 confocal microscope (Carl Zeiss, Jena, Germany).