Bx60 microscope

Double immunofluorescent labeling for TRPV1-neurofilament 200 was performed using polyclonal rabbit anti-rat TRPV1 antibodies, 1:1000 (Affinity Bioreagents, Golden, CO, USA) and mouse monoclonal antibody N52 (1:2000) to the C-terminal fragment of neurofilament 200 (Chemicon International, Temecula, CA, USA). After deparaffinization, antigen retrieval and 2 washes in PBS, sections were incubated in blocking solution (10% normal horse serum and 0.1% Triton X-100 in PBS) for 2 hours. The 2 primary antibodies were mixed in DAKO protein block solution and incubated overnight at 4 °C. Sections were rinsed 3 times in PBS followed by another 20-minute block and then addition of Alexa Fluor 594 donkey anti-mouse and Alexa Fluor 488 donkey anti-rabbit secondary antibodies (Invitrogen), for TRPV1 and NF200, respectively, in blocking solution and incubated for at least 2 hours. After three 5-minute washes with PBS, the slide-mounted sections were cover-slipped with Fluoromount-G (Southern Biotech, Birmingham, AL, USA). Images were acquired on an Olympus BX-60 microscope equipped with a Hamamatsu ORCA-ER camera (Hammamatsu City, Japan) using separate band-pass filters, then merged. Counts were obtained from sections of paraffin embedded DRG with a 20− objective. We counted 22 fields from 3 sections in 3 rats, for a total of 1086 counted DRG cells (Fig. 6).

For immunofluorescence microscopy, cells were grown to mid-log phase at the indicated temperatures and fixed using paraformaldehyde and glutaraldehyde as previously described (Levin, 2002 ). Briefly, 500 µL of mid-log phase cells were fixed in paraformaldehyde and glutaraldehyde for 15 minutes at room temperature followed immediately by incubation for 30 minutes on ice. Cells were washed 3 times with 1X PBS and resuspended in 1X GTE. FtsA was stained using 1:500 anti-FtsA primary antibody and 1:200 secondary anti-rabbit IgG conjugated to Alexa Fluor 488 (Life Technologies) or DyLight 550 (Pierce). Anti-FtsZ was stained with 1:2000 anti-FtsZ primary antibody and 1:200 of the Alexa Fluor 488 or DyLight 550 conjugated goat-anti-rabbit IgG secondary antibody. Fluorescence images were captured using an Olympus BX60 microscope with a 100X oil immersion objective and a Hamamatsu C8484 digital camera with HCImage software. Images were compiled using Adobe Photoshop.