Rabbit anti hcn2

Rats from each group were sacrificed on 21 d, followed by isolation of the L3-5 DRG and storage in liquid nitrogen or at −80°C for subsequent procedures. The frozen DRG were directly homogenized in a lysis buffer containing a cocktail of protease inhibitors. Equal amounts of protein (30 μg) were loaded in each lane and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The membranes were blocked in 3% bovine serum albumin (BSA) for 2 h and then rinsed thrice in washing buffer for 5 min each. Following sequential incubation overnight at 4°C with rabbit anti-HCN2 (1 : 200, Alomone Labs) and mouse anti-β-actin (1 : 1000) primary antibody, the blots were rinsed thrice in washing buffer for 5 min each and incubated for 2 h with AP-conjugated goat antirabbit IgG (1 : 1000, Beyotime Biotechnology, Nantong, China) or goat antimouse secondary antibody IgG (1 : 1000). All western blot analyses were performed at least in triplicate, with parallel results obtained. Squares of the same size from each band were measured for density, with the background subtracted. The expression of β-actin was used as a loading control for protein expression. The expression level of the proteins is an average of the densities per band area from each group.

On 21 d after STZ injection, rats were anesthetized with pentobarbital sodium and transcardially perfused with normal saline succeeded by 4% paraformaldehyde in 0.1 M phosphate buffer saline (PBS). DRGs from L3-5 were isolated, postfixed overnight in 4% paraformaldehyde in 0.1 M PBS, and cryoprotected in 30% sucrose in 0.1 M PB at 4°C till the tissue sinkage to the bottom of the container. Transverse DRG sections (14 μm) were sliced on a cryostat and mounted serially on slides. To visualize HCN2 channels, immunofluorescence labeling methods were used. Briefly, the sections were rinsed twice with 0.1 M PBS and then incubated with a solution containing 0.3% Triton X-100 and 1% donkey serum albumin for 3 h at r/t. The sections were thereafter incubated with anti-HCN2 channel antibodies (1 : 200, rabbit anti-HCN2, Alomone Labs) in PBST for 24 h at 4°C. After three washes with PBST, the sections were further incubated with secondary antibody Alexa Fluor 488 (1 : 200, donkey antirabbit IgG, Invitrogen, USA) for 2 h at r/t. The sections were then mounted with antifade medium and stored at 4°C. All images were captured with Olympus FV1000 confocal microscope and processed with Adobe Photoshop software. The negative controls were performed using the same procedure without addition of primary antibody.

DRG neurons were fixed in 4% paraformaldehyde in PBS for 15 min and permeabilized in 0.3% Triton X-100-PBS for 10 min. Cells were then blocked in 1% BSA for 10 min and incubated with rabbit anti-HCN1 [1:300], rabbit anti-HCN2 [1:100], rabbit anti-HCN3 [1:25], rabbit anti-HCN4 [1:200] (Alomone Labs, Israel) antibodies overnight at 4°C and Alexa Fluor 546 anti-rabbit (Invitrogen) secondary antibodies for 2 h. To spot nuclei, the sample was incubated with, 4′, 6-diamidino-2-phenylindole [1:1000] (DAPI, Vectashield Labs, UK) in 0.1% Tween 20 in PBS for 10 min. Images were obtained using a fluorescence microscope (Olympus BX63, Italy) with a 20X objective and a CellSens Dimension Imaging Software (Olympus, Italy). HCN immunofluorescence in cultured DRG neurons was semi-quantitatively measured on a computer using ImageJ 1.33 image analysis software (http://rsb.info.nih.gov/ij), as described in Bigagli et al. (2018 (link)). Briefly, eight photomicrographs were randomly taken of each sample and for each cell total, membrane, or cytoplasmic HCN fluorescence was measured and expressed as pixels. These values were used to calculate the membrane/cytoplasmic relative fluorescence (% total cell fluorescence) of HCN channels.