Alexa fluor 555 conjugated donkey anti mouse

Primary antibodies against P62 (pm045), and LC3 (pm036) were purchased from MBL (Japan). An antibody against KEAP1 (#7705) was obtained from Cell Signaling Technology (Beverly, MA). An antibody against NRF2 (sc-722) was purchased from Santa Cruz. An anti-αSMA antibody (ab7817) was obtained from Abcam. Alexa Fluor 488-conjugated goat anti-rabbit, Alexa Fluor 555-conjugated donkey anti-mouse, HRP-conjugated goat anti rabbit, and HRP–conjugated rabbit anti-mouse antibodies, all primers for gene expression analyses, and Lipofectamine RNAiMAX reagents were purchased from Invitrogen. tBHQ (tertiary butylhydroquinone), anti-β-actin, DMEM (high glucose), and 2× Laemmli sample buffer were purchased from Sigma.

Immunofluorescent labeling of the following markers was performed on different group rat lumbar DRG cryosections using the methods as previously described [16 (link)]. The primary antibodies in the immunohistochemistry: rabbit polyclonal and mouse monoclonal antibodies against pCaMKIV, HMGB1, isolectin B4 (IB4), protein gene product 9.5 (PGP9.5), calcitonin gene-related peptide (CGRP), 1:200, Santa Cruz. The secondary antibodies used: Alexa Fluor 555-conjugated donkey-anti-mouse, 1:500; Alexa Fluor 488-conjugated donkey-anti-rabbit, 1:500, Invitrogen.
The cells were visualized using a laser confocal microscopic imaging system (Nikon A1; Nikon Co.Ltd., Tokyo, Japan) with a water immersion 10 objective lens (Plan Apo 60x1.20 PFS WI) and a 1 st dichroic mirror (405/488/561/640). The green signal was excited by 488 nm light from an Ar laser and red signal was excited by 561 nm light from a DPSS laser. The images were 400 times magnification and the scale bar in Fig. 4 was 20 μm.