Goat anti mouse secondary antibody

After fixation, tumor tissues were embedded in paraffin, and tissues sections were cut at a 5-μm thickness and rehydrated in xylene (Sigma Cat. no.: 247642) and descending concentrations of 75% ethanol for 5 min, and then washed 4 times using PBS (5 min for each wash). Sections were incubated in 3% horse serum to block nonspecific binding. Sections were then incubated with anti-CD206 (Proteintech, Cat. no.: 18704-1-A P) and anti-CD31 (Affinity Biosciences, Cat. no.: AF6191 antibodies overnight at 4°C. Primary antibodies were detected by appropriate secondary antibodies; e.g., goat anti-rabbit secondary antibody (1:200; Servicebio, Cat. no.: GB23303) or goat anti-mouse secondary antibody (1:200; Servicebio, Cat. no.: GB23301).

All the tissues were sliced with microtome at 4 μm thickness. To detect meniscus cells (chondrocytes) in the neo-meniscus and analyze collagen distribution, matrix stainability and matrix contents, glycosaminoglycan, H&E staining, Masson’s trichrome and Safranin-O staining were used. For toxicity analysis, H&E staining was used to examine the morphology of key organs. Immunostaining for type I & II collagen in the regenerated meniscus was carried out to show the expression and distribution of collagen. Sections were incubated with the following primary antibodies: Collagen I Antibody (Col-1) (1:100; GeneTex, GTX26308), Anti-Collagen Type II (Ab-1) mouse mAb (II-4C11) (1:200; Sigma-Aldrich, CP18-100UG). Immunostainings were finished using Goat anti-mouse secondary antibody (1:1000; Servicebio, G1214).
For quantification of histology for regenerated meniscus, Pauli’s scoring system was used as the previous report,^{41 (link),42 (link)} and this scale assesses different aspects of the tissue histology including regenerated tissue surface, cellularity, collagen fiber organization, and matrix stainability with safranin-O. After staining, all sections were photographed under a microscope (Hamamastu, NanoZoomer S360).

The frozen spinal cord L4–6 was ground and mixed with cold RIPA Buffer (200 µL per 20 mg of tissue) (Beyotime Biotech, SH, China). Forty micrograms of proteins were subjected to 10% SDS-PAGE and transferred to a PVDF membrane using a minigel and mini transblot apparatus (Bio-Rad, Hercules, CA, USA). After being incubated in 5% nonfat milk containing 0.1% Tween-20 for 2 h, the membrane was infiltrated with the primary mouse anti-Syt-1 antibody (4 µg/mL, R&D Systems, Minneapolis, MN, USA) at 4 °C overnight. The membrane was washed in Tris-Buffered saline Tween-20 (TBST) 3 times and incubated in the goat anti-mouse secondary antibody (1:3000, Servicebio, WH, China) at 37 °C for 1 h. GAPDH (1:1000, Servicebio, WH, China) was used as the protein control. The antigen-antibody complex was reacted with a horseradish peroxidase substrate (Millipore, Burlington, MA, USA) and visualized using the ImageQuant LAS 4000 min CCD camera (GE Healthcare, Boston, MA, USA). The bands were analyzed by Quantity One software (Bio-Rad, Hercules, CA, USA). Values of Syt-1 were represented as the optical density ratio of the target protein bands to the related GAPDH bands.