Rhodamine conjugated goat anti rabbit igg

Sections were prepared from fresh frozen brains taken 48 hours post-SAH. Sections were incubated with a rabbit monoclonal anti-IBA1 antibody (microglial marker, 1:100; Proteintech Group, Inc.) for 16 hours at 4°C, followed by a secondary antibody (rhodamine-conjugated goat anti-rabbit IgG, 1:100; Proteintech Group, Inc.) for 30 minutes at 37.4°C and then DAPI for 10 minutes at room temperature (Table 1). Images were captured and analyzed using fluorescence microscopy (BX51; Olympus, Tokyo, Japan) with the Magna Fire system. Six images at 400× magnification (n = 4) were randomly selected for each slice, and the results were analyzed and quantified using the Image-Pro Plus 6.0 image analysis system (Media Cybernetics). The number of positive cells in each section is expressed as the average of six images per section. The results are expressed as a ratio of positive cells and compared with the sham group.

Rats were deeply anesthetized with pentobarbital (100 mg/kg, i.p.) and then underwent transcardiac perfusion of 150 ml of PBS containing 0.1% heparin, followed by 500 ml of ice-cold 4% paraformaldehyde. Next, the lumbar enlargements were harvested, fixed in 4% paraformaldehyde for 8 h, and cryoprotected in 30% (w/v) sucrose. Serial frozen sections embedded in medium (Tissue-Tek O.C.T. Compound, Torrance, CA, USA) were cut to obtain 10 µm of thickness with CM1800 (Leica, Heidelberg, Germany). All sections were rinsed in 0.3% Triton X-100 for 15 min, and then blocked with 10% goat serum for 1 h at 37°C, before incubation with primary antibodies overnight at 4°C at the following dilutions: antimouse GFAP (1:300; Cell Signaling, MA, USA), antirabbit BMP2 (1:200; Abcam, Cambridge, UK), antirabbit BMP4 (1:200; Abcam), and antirabbit p-STAT3 rabbit IgG (1:100; Cell Signaling). This was followed by incubation for 2 h at room temperature with a mixture of fluorescein isothiocyanate-conjugated goat antimouse IgG (1:150; Proteintech, IL, USA) and rhodamine-conjugated goat antirabbit IgG (1:150; Proteintech). The tissue sections were then washed and subsequently incubated with 4′,6-diamidino-2-phenylindole for 15 min. Finally, all sections were evaluated with fluorescence microscopy (Nikon eclipse E600, Japan).