Goat anti mouse igg conjugated with alexa 488

For tryptase staining in the stomach, paraffin-embedded tissue sections were used. After deparaffinizing, sections were washed with PBS for 5 minutes, 3 times, followed by blocking with 10% normal goat serum (NGS) and 5% BSA in PBS in 1 hour. The sections were then incubated overnight with mouse anti–mast cell tryptase (1:300, NBP2-26444, Novus Biologicals) and rabbit anti-PGP9.5 (1:400, Z5116 DAKO/Aligent) in PBS containing 5% NGS, 1 % BSA, and 0.1% Triton X-100. After 3 washes with PBS, sections were incubated with goat anti–mouse IgG conjugated with Alexa 488 and goat anti–rabbit IgG conjugated with Alexa 594 (1:500, A-11001 and A-11012, respectively; Thermo Fisher Scientific) for 2 hours. After the wash, the slides were mounted with ProLong Gold Antifade Mountant solution (P36931, Thermo Fisher Scientific).
For FOS staining, the spinal cord (T8–T10) was collected and sectioned to 14 μm slices that were mounted on slides. The immunostaining procedure was similar to that for tryptase, except goat anti-FOS (sc271243, Santa Cruz Biotechnology Inc.) at 1:100 dilution was used.
The images were captured by a fluorescent microscope with 20× lens. The images were analyzed using ImageJ (NIH) software. Four to 6 sections from each animal and 4–5 rats in each group were analyzed.

Expression of GPR34 mutants was measured by flow cytometry. In brief, HEK293A cells were transfected with human GPR34-encoding plasmid (250 ng), using polyethyleneimine. Cells were then suspended in 200 ml of D-PBS, containing 2 mM EDTA, and dispensed into 96-well V-bottom plates. After centrifugation for 1 min at 700 × g, the cells were suspended in 200 ml/well of FACS buffer (D-PBS, containing 0.5% BSA and 2 mM EDTA) and incubated for 30 min on ice. Cells were then centrifuged again for 1 min at 700 × g, resuspended in 25 ml/well of anti-human GPR34 serum (1/100 diluted), and incubated for 30 min on ice. After centrifugation for 1 min at 700 × g, cells were washed with D-PBS, resuspended in 25 ml/well of goat anti-mouse IgG conjugated with Alexa488 (Thermo Fisher Scientific, 10 mg/ml), and incubated for 15 min on ice. Cells were then centrifuged a final time for 1 min at 700 × g, washed with D-PBS, and resuspended in 150 ml/well of D-PBS, containing 2 mM EDTA. Flow cytometry analysis was performed with the BD FACSLyric Flow Cytometry System (BD Biosciences, Franklin Lakes, NJ, USA), and the data were analysed by FlowJo Software (FlowJo, Ashland, OR, USA).

Plasmids, specifically pReceiver-M61C-LAMC2, were constructed by GeneCopoeia (USA) and amplified at the School of Basic Medical Science, Zhejiang University. Chitosan (CS, molecular weight, 100,000) was purchased from Qingdao Yunzhou Biochemistry (China). Fluorescein-isothiocyanate-labeled phalloidin, Triton X-100, CelLytic buffer, and hyaluronic acid (HA) were purchased from Sigma Chemical Co. (MO, USA). A Label IT^® TM-Rhodamine labeling kit was purchased from Mirus Bio, LLC. (USA). Alamarblue and Lipofectamine LTX Plus reagent were purchased from Invitrogen (USA). Bovine serum albumin was purchased from Shanghai Sangon Biological Engineering Technology and Services (China). Hoechst 33258 DNA dye was purchased from Beyotime (China). Anti-laminin γ2 mouse monoclonal antibody, anti-integrin β4 mouse monoclonal antibody and anti-plectin rabbit monoclonal antibody were purchased from Abcam (USA). Goat anti-mouse IgG conjugated with Alexa 488 and goat anti-mouse IgG conjugated with Alexa 594 were purchased from Invitrogen (USA). Goat anti-rabbit IgG conjugated with rhodamine was purchased from Jackson ImmunoResearch Laboratories (USA). A human laminin-5 ELISA kit was purchased from R&D Technologies. Flat pure titanium plates 10 × 10 × 1 mm in size were purchased from Zhejiang Guangci Medical Appliance Company (China).