Goat anti rabbit igg secondary antibody

Interlaminar ligamentum flavum, interspinous ligament, or supraspinal ligament tissue was obtained from AS patients and traumatic injury patients who had spinal fractures when undergoing spinal surgery as controls. The tissues were fixed in 4% paraformaldehyde for 4–6 h and then embedded in paraffin. The paraformaldehyde-fixed paraffin-embedded sections were cut into 5 μm using a Leica RM2235. Paraffin-embedded sections were first deparaffinized twice in a series of 100% xylene, rehydrated in a series of graded ethanol (100%, 100%, 95%, and 80%), and then washed briefly in distilled water and PBS, respectively. Antigen thermal repair was performed with 0.01 M citrate buffer (pH6.0) or EDTA buffer (pH9.0) at high pressure, washed in PBS, sections were treated with 3% hydrogen peroxide-methanol for 10 min and blocked with normal goat serum for 30 min. Then, the sections were incubated overnight with PDGFB (1:100, Abcam, catalog: ab23914); GRB2 (1:50, Abcam, catalog: ab32037); P-ERK (1:100, Abcam, catalog: ab278538); RUNX2 (1:50, Abcam, catalog: ab76956) antibody at 4 °C. Goat anti-rabbit IgG secondary antibody (JACKSON, catalog: 111–035-003) was incubated and DAB solution (Sigma, catalog: D8001) was used for color development. All images were obtained using an Open-field slice scanner (NanoZoomer S210).

Very small pieces (approximately 1 mm³) of #4 mammary glands were fixed in 2% glutaraldehyde and 1% paraformaldehyde overnight. They were then fixed again in OsO4 for 2 h before embedding using the Embed 812 Kit (Electron Microscopy Sciences, Cat# 14120). Ultrathin sections were cut using a diamond knife and double-stained with uranyl acetate and lead citrate before the examination.
For immuno-electron microscopy, tissues were fixed in 0.2% glutaraldehyde and 4% paraformaldehyde before they were embedded in Epon 812. Ultrathin sections were then cut and sequentially treated with sodium periodate, 0.1% glycine, and 1% BSA before they were incubated with OCLN antibody for 48 h at 4°C. After washing, samples were incubated with goat anti-rabbit IgG secondary antibody, which was conjugated with 10 nm gold particles (Sigma, #G7402) at a 1:40 dilution for 2 h at RT. Samples were refixed with 1% glutaraldehyde, double-stained with uranyl acetate and lead citrate, then examined using a Joel JEM-1230 (Institute of Neurosciences, Academia Sinica) or a Talos L120C (ShanghaiTech University) transmission electron microscope at an accelerating voltage of 80 kV.

The intestinal tissues were washed with cold PBS and lysed with lysis buffer (Beyotime, China). The protein quantity was detected using the BCA protein assay Kit (Beyotime, China). Proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Germany). Then, the membranes were blocked with 5% BSA in Tris-buffered saline containing 0.2% Tween-20 (TBST) for 2 h at room temperature, washed extensively with TBST and incubated with the primary antibody: rabbit anti-GPR41 (Cohesion, United Kingdom), rabbit anti-GPR109A (Cohesion, United Kingdom), mouse anti-GLP-1 antibody (Abcam, United States), rabbit anti-GAPDH antibody (Proteintech Group, United States), mouse anti-tubulin antibody (Ray Antibody Biotech, China) at 4°C overnight. Finally, the membranes were incubated with secondary antibody: goat anti-rabbit IgG Secondary Antibody (Sigma, United States) or goat anti-mouse IgG Secondary Antibody (Invitrogen, United States) and the blots were developed with a chemiluminescence reagent (Millipore, Germany).

Wild type and mutant Rpph1 sequences were cloned into pcDNA4a (Invitrogen) and psiCHECK2 (Promega) vectors. MREs of miR-326-3p (5′-CCCAGAG-3′) and miR-330-3p (5′-CCCAGAGA-3′) on Rpph1 were mutated into 5′-GCACAGAC-3′. All plasmids were prepared in endotoxin-free conditions by QIAGEN Plasmid Midi Kit (Qiagen) or Tiangen Plasmid Mini Kit (Tiangen Biotech., Beijing, China). H₂O₂ was from the Guangzhou chemical reagent factory, Guangzhou, China. Glucose and Aβ 1–42 were purchased from Sigma Aldrich. MiR-326-3p and miR-330-3p mimics were purchased from RiboBio, Co., Ltd. (Guangzhou, China). Rabbit polyclonal antibodies against CDC42 and β-actin were from Abcam (ab187643) and Cell Signaling Technology (4970), respectively. The secondary goat anti-rabbit IgG antibody (A9169) was from Sigma.