Alexa 488

At 16 weeks after surgery, the tibial nerve was harvested after carbon dioxide euthanasia. The nerve was treated with 4% paraformaldehyde for a duration of 24 h, followed by slicing the nerve into transverse sections of 12 μm thickness using a frozen slicer. Immunofluorescence staining was conducted using an established protocol with NF200 (MilliporeSigma, Burlington, MA, USA) staining targeted toward monitoring axonal regeneration and S100 (MilliporeSigma, Burlington, MA, USA) staining to detect Schwann cells. In short, sections of nerve tissue were incubated with primary antibodies that targeted NF200 and S100 for an overnight period at 4 °C, followed by rinsing with PBS. Afterwards, secondary antibodies conjugated with Alexa488 (Abcam, Cambridge, UK) and Alexa594 (Abcam, Cambridge, UK) were added and incubated at room temperature for 1 h. DAPI (Beyotime, Shanghai, China) was applied to the nucleus and incubated for 5 min. The tissue sections were observed via a 3D fluorescence imaging system (HOOKE INSTRUMENTS, Changchun, China).

Ten μm-thick coronal brain sections taken on a cryostat (CM3050; Leica; Buffalo Grove, IL, USA) were used for immunofluorescence analysis to examine galanin (1:200 dilution, T-4334; Peninsula Laboratories T-4334; San Carlos, CA, USA) immunoreactivity in the hippocampus. Tissues were washed with PBST (PBS + 0.1% Triton X100) prior to antigen retrieval with 10 mM sodium citrate (pH 6.0). Tissues were blocked in 3% PBST (PBS + 3% BSA + 0.4% Triton X100) for one hour before being incubated with the primary antibody overnight in a humidified chamber at 4°C. The next day tissues were washed with PBST then incubated with the secondary antibody (1:500, Alexa 488; Abcam; Cambridge, MA, USA) and DAPI (1 mg/ml diluted 1:500; Thermo Scientific; Waltham, MA, USA). Slides were washed again and coverslips were mounted with glycerol (G7893, 70% in water; Sigma-Aldrich; St. Louis, MO, USA). Images were captured on an Olympus IX81 Motorized Inverted Fluorescent Microscope (Center Valley, PA, USA) and quantified by calculating corrected total cell fluorescence [CTCF = Integrated density − (area of selected cell × mean fluorescence of background readings)] using Image J software (NIH; Bethesda, MD, USA).

Nerve segments were prepared for immunocytochemistry by blocking in 3% normal goat serum for 30 min at 37 °C. Permeabilized fibers were incubated with anti-BrU (Sigma, 1 : 300) overnight at 4 °C. Fibers were washed 6 times 5 min each. Secondary antibodies (goat anti-mouse or goat anti-rabbit conjugated with Alexa 488 (Abcam, 1 : 1000) were incubated for 45 min at 37 °C. F-actin was detected using fluorescent phalloidin (Invitrogen) added together with secondary antibodies. Fibers were then washed six times 5 min each. Finally, individual fibers were teased and mounted in ProLong Antifade (Invitrogen).

The dewaxed tissue sections were alternately placed in citric acid buffer and heated to boiling point for antigen repair for 20 min, and then returned to room temperature. The cells were permeated with PBS which contained 0.3% Triton X-100 for 20 min. These slices were closed with 3% Donkey serum and incubated in an incubator for 30 min. The specific fluorescent antibody was incubated overnight at 4°C and the fluorescent secondary antibody was incubated for 1 h. Primary antibodies anti-F4/80 antibody (1/100, abcam), anti-iNOS antibody (NOS2) (1/100, abcam), and anti-CD206 (MMR) monoclonal antibody (MR6F3)-PE (1/50, invitrogen) were utilized. For anti-F4/80, anti-iNOS and anti-CD206 staining, donkey anti-rabbit (1/500, Alexa 488,abcam), goat anti-rabbit (1/500, Alexa 647,Invitrogen), and donkey anti-goat (Alexa Fluor^®555) secondary antibodies were used. After antibody incubation, anti-fluorescence quenching sealing tablets (including DAPI) were added to seal the tablets. Images were collected and analyzed by laser confocal microscopy system (TCS SP8 X, Leica). Immunofluorescent antibody markers were matched with protocols provided in the relevant literature (Yu et al., 2016 (link)). F4/80⁺CD206⁻iNOS⁺ cells were M1-type macrophages, and F4/80⁺CD206⁺ iNOS^-cells were defined as M2-type macrophages.

BV2 cells were cultured on 24-well plates and transfected as described above. 24 h post-transfection, cells were stimulated with 1000 ng/ml LPS for 12 h, followed by collection of bright-field images using a Nikon microscope. The cells were then rinsed with PBS, fixed in 4% paraformaldehyde in PBS at RT for 10 min, and permeabilized with 0.1% Triton X-100 in PBS for 10 min. The cells were subsequently blocked in 2% bovine serum albumin in PBS at RT for 1 h, followed by incubation with a goat anti-Iba1 monoclonal primary antibody (1:500, Abcam, United States) at 4°C overnight. The following day, the cells were washed 3 times with PBS, and incubated sequentially with a donkey anti-goat IgG secondary antibody Alexa 488 (1:1000, Abcam, United States) at RT for 2 h, the cytoskeleton red fluorescent probe ActinRed (1:50, KeyGEN BioTECH, China) at RT for 20 min, and DAPI at RT for 5 min, and finally washed 3 times with PBS. Fluorescence intensity was detected using a Zeiss LSM710 fluorescence microscope.

Coronal sections were prepared on a cryostat at 20 μm and immediately mounted on slides. Slides were maintained at −80°C until ready for staining. Every 10th section was Nissl stained with Cresyl Violet to identify sections corresponding to areas of interest (Franklin and Paxinos, 2013 ).
Immunostaining was done as described previously (Beaudoin et al., 2012 (link)). The following primary antibodies were used mouse anti-bassoon (1:400, ABCAM ab82958), rabbit anti-gephyrin (1:1000, ABCAM ab32206), rabbit anti-N-methyl-d-aspartate (NMDA) receptor subunit 1 (1:1000, Thermo Fisher Scientific PA3-102), and chicken anti-tyrosine hydroxylase (TH) (1:500, ABCAM ab76442). Secondary antibodies used were goat anti-mouse (1:1000 for all; Alexa568, ABCAM ab175473; Alexa488, Fisher A11029; Alexa647, Fisher A21241), goat anti-rabbit (1:1000 for all; Alexa488 ABCAM ab150077; Alexa568, Fisher A11036), and/or goat anti-chicken (1:1000, Alexa405, ABCAM ab175675). Coverslips were mounted with ProLong Gold antifade (Invitrogen).