Ab75810

Total protein from the spinal cord tissue was purified using protein extraction reagents containing 1% protease and phosphatase inhibitors. The protein concentration of each sample was quantified with Carmassi Bradford reagents (Thermo). An equivalent amount of protein (60 µg) was separated by 10% SDS‐PAGE and transferred onto PVDF membranes (Bio‐Rad). After blocking with 5% (w/v) non‐fat milk, the membranes were further incubated with primary antibody solutions overnight at 4°C. The following primary antibodies were including: TrkA (ab‐76291, Abcam, 1:5000), FGFR1 (ab‐58516, Abcam, 1:1000), P‐AKT (sc‐514032, Santa Cruz, 1:1000), AKT (sc‐81434, Santa Cruz, 1:1000), P‐ERK (sc‐16982, Santa Cruz, 1:1000), ERK (sc‐514302, Santa Cruz, 1:1000), Bcl‐2 (60178‐1‐Ig, proteintech, 1:3000), Bax (60267‐1‐Ig, proteintech, 1:2000), cleaved caspase‐3 (sc‐373730, Santa Cruz, 1:500), GAP43 (ab75810, Abcam, 1:10 000), GFAP (ab7260, Abcam, 1:2000), NF‐200 (ab8135, Abcam, 1:5000) and GAPDH (K200057M, Solarbio, 1:5000). After three washed with TBST, the membranes were incubated with a 1:10 000 dilution of horseradish peroxidase‐conjugated secondary antibodies for 60 minutes at room temperature. Finally, signals were visualized by Chemi DocXRS + Imaging System (Bio‐Rad). All experiments were repeated three times.

Primary antibodies including neurofilament 200 (NF200, ab4680), glial fibrillary acidic protein (GFAP, ab7260), NeuN (ab104224), growth associated protein 43 (GAP43, ab75810), and LC3-II (ab192890), and secondary antibodies including goat anti-mouse 488 (ab150113), goat anti-rabbit 488 (ab150077), goat anti-chicken 488 (ab150169) and goat anti-rabbit tritc (ab6718) were purchased from Abcam (MC, United Kingdom). The DAPI was also under the supply of Abcam (MC, United Kingdom). Recombinant human FGF21 (rhFGF21) was obtained from Prof. Xiaokun Li, Zhejiang Provincial Key Laboratory of Biopharmaceuticals, Wenzhou Medical College, Wenzhou, Zhejiang, China. It was previously reported that rhFGF21 was produced using Escherichia coli and purified to be endotoxin free (Wang et al., 2010 (link)), and its biological effect further tested in nerve cells (Lu et al., 2019 (link)).

1 mL RIPA lysis buffer solution was added to H9c2 cell and tissue samples for homogenization. Samples were lysed on ice for 30 minutes for protein extraction, and BCA protein assay kit was utilized to quantify the obtained protein. The total amount of loaded protein was 40 ug, 20 ul. The protein was separated on a gel prepared with an appropriate percentage to detect the molecular weight of the target protein and then followed by electrophoresis and electrotransformation. The PVDF membrane after electroporation was immersed in a TBS-T blocking solution containing 5% milk for sealing. Then, it was incubated overnight combined with NGF antibody (ab52918, Abcam), GAP43 antibody (ab75810, Abcam), Semaphorin 3A antibody (ab11370, Abcam), Tyrosine Hydroxylase antibody (ab112, Abcam), ERK antibody (9102, CST), p-ERK antibody (9101, CST), c-fos antibody (ab7963, Abcam), c-Myc antibody (ab39688, Abcam), and actin antibody (ab8226, Abcam). The relative expression level of protein was detected by electrochemiluminescence and gray-scale scanning gel imaging analysis.

Spinal cord sections were subjected to xylene dewaxing, gradient alcohol rehydration, and antigen retrieval with boiling 0.01 M citrate buffer. Endogenous peroxidase activity was eliminated by 15 min of incubation with 3% hydrogen peroxide. Then, these sections were incubated with goat serum blocking solution for 15 min at room temperature and incubated with primary antibodies (rabbit anti-rat NF200, ab8135, GFAP, ab7260, GAP-43, ab75810, 1:100, Abcam, UK) for 12 h at 4°C. After washing 3 times with PBS, sections were incubated with horseradish peroxidase-labeled goat anti-rabbit IgG (1:500, Beijing Zhongshang Jinqiao Biotechnology Co., Ltd.) for 15 min at 37°C. Followed by DAB color reaction and hematoxylin counterstaining, sections were sealed with neutral gum and observed under an optical microscope (Olympus). Positive cells were counted in 5 random fields of view.

The sample sectioning procedures and details of the staining techniques were performed as previously described. In order to detect nerve fibers in the heart, antibodies against nerve growth factor (NGF, AB52918, 1:1,000; Abcam, Cambridge, UK) and growth associated protein 43 (GAP43; AB75810, 1:1,000; Abcam) were used. An image processing system was used to quantitatively detect the NGF- and GAP43-positive regions through the use of immunohistochemical stains, and these values were expressed as the average optical density (AOD).
A computer-assisted morphometric analysis system (Image-Pro Plus 6.0) was used to determine nerve density. The computer automatically recognized the color of the immunohistochemical stains and calculated the pixel area of the nerves. Each slice was observed at 40× under a microscope. The image was saved at a resolution of 4080×3072 pixels. One to three sections from each animal were observed, and three maximum and minimum nerve density fields from each section were selected. The average density of the maximum nerve density fields was taken as the average density of a section. The average difference between the maximum and minimum nerve density fields was defined as the heterogeneity of the local nerve distribution.

Equal amounts of protein (50 μg) were separated on 8–16% Tris-glycine gels (Novex, Invitrogen, CA, USA) and transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with 5% skim milk, the PVDF membranes were incubated overnight at 4°C with the following primary antibodies: rabbit polyclonal anti- NGF antibody (AB52918, 1:1,000; Abcam), anti-GAP43 antibody (AB75810, 1:1,000; Abcam) and mouse monoclonal anti-GAPDH antibody (1:2,000; Abcam). On the next day, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies with appropriate species specificity. Immunoreactivity was visualized using an enhanced chemiluminescence (ECL) detection kit (LAS-4000 mini, Fujifilm, Japan). The immunoreactive bands were quantified by densitometry using the LAS-4000 mini 2200 software (Fujifilm) to estimate the protein expression. All protein expression signals were normalized to the intrasample GAPDH signal.

Spinal cord tissue, or neurons were fixed in 4% PFA for 20 min. Thenwe were incubated with primary rabbit anti‐SRF antibody (1:200 diluted by PBS, CST, D71A9, USA), anti‐Ras antibody (1:500 diluted by PBS, Abcam, ab52939, USA), anti‐Raf antibody (1:250 diluted by PBS, Abcam, ab33899, USA), anti‐cofilin antibody (1:300 diluted by PBS, Abcam, ab42824, USA), anti‐GAP43 antibody (1:500 diluted by PBS, Abcam, ab75810, USA) and anti‐NF antibody (1:50 diluted by PBS, ZSGB‐BIO, 18703E05, China) overnight at 4°C, incubator with DAPI (1:700 diluted by PBS, Solarbio, China) 10 min, and washed with PBS for 5 times (3 min each time). After washing, the secondary antibody (1:500, Abcam, USA) was applied, and the mixture was incubated at 37°C for 1 h. Images were recorded using a microscope.