Alexa fluor 594 conjugated goat antirabbit igg secondary antibody

Half of the mice were anesthetized and perfused transcardially with PBS, followed by 4% paraformaldehyde. The brain was then postfixed in 4% paraformaldehyde overnight. Brain sections were embedded in paraffin, cut to 4-μm thickness. The paraffin-embedded tissue cross-sections were dewaxed in xylol, rehydrated, antigen retrieval, washed with PBS, and blocked with 1% BSA (Roche, Switzerland) in PBS for 2 h at room temperature. The sections were incubated at 4 °C overnight with primary antibodies against rabbit anti-CRMP2 (1:500, Abcam), rabbit anti-Iba1 (1:400, CST) and mouse anti-α-tubulin (1:1000, Abcam), followed by incubation with Alexa Fluor 594-conjugated goat antirabbit IgG secondary antibody (1:250, Abcam) and Alex Fluor 488-conjugated goat antimouse IgG secondary antibody (1:200, Abcam) for 120 min. 4’,6-diamidino-2’-phenylindole (Thermo Fisher, Massachusetts, USA) was used as a nuclear stain. The images were collected under an inverted fluorescence microscope (IX53, Olympus, Tokyo, Japan). Image J was used to analyze the integral optical density of the target protein.

As described previously,^{44 (link)} osteoblast adhesion and morphology were assessed
by visualizing the intracellular actin filaments and nucleus following
staining with rhodamine phalloidin (Millipore Sigma, USA) and DAPI
(Millipore Sigma, USA), respectively. The osteogenic differentiation
potential was detected by ALP staining using BCIP/NBT ALP color development
kit (Beyotime Biotechnology, China) following incubation for 7 days
under osteogenic conditions. The ROS levels were determined by staining
the cells using a DCFH-DA fluorescence kit (Beyotime Biotechnology,
China). Immunofluorescence staining was carried out using primary
antibodies against NRF2 (1:200 dilution; Abcam, UK), an Alexa Fluor
594-conjugated goat antirabbit IgG secondary antibody (1:400; Abcam),
and DAPI for nuclear staining. All the above staining procedures were
carried out as per the manufacturers’ instructions, and the
images were visualized and captured using light (Olympus IX73, Olympus
Lifescience, Japan) or fluorescence (Leica, Germany) microscopes.