Goat anti rabbit igg

Mouse testicular and epididymal samples were collected, washed three times with PBS, fixed in 4% paraformaldehyde at 4 °C overnight, embedded in paraffin, and sliced into 5 μm sections (Leica, Wetzlar, Germany). After dewaxing and rehydration, the prepared sections were repaired in citric acid antigen retrieval solution (Servicebio, Wuhan, China) at 96 °C for 10 min and cooled naturally to room temperature. Then, the sections were washed with prewarmed Tris Buffered Saline (TBS;Servicebio, Wuhan, China), blocked with QuickBlock™ Blocking Buffer (Beyotime, Shanghai, China) for Immunol Staining for 10 min, and incubated with the primary antibody overnight at 4 °C. The primary antibodies used were a Recombinant Anti-CD63 antibody (dilution 1:100, #Ab217345; Abcam, Cambridge, UK) and SMMHC/MYH11 Rabbit pAb (dilution 1:100, #A10827; ABclonal, Wuhan, China). After incubation, the sections were washed three times with TBST (Servicebio, Wuhan, China) and then incubated with secondary antibody for 30 min at 37 °C. Goat Anti-Rabbit IgG (dilution 1:50, #AS060; ABclonal, Wuhan, China) was used as the secondary antibody. Cell nuclei were labelled with Antifade Mounting Medium containing DAPI (dilution 1:1000; Beyotime, Shanghai, China).

Protein was extracted from four pairs of tissue from HCC patients using RIPA buffer (Pierce, Rockford, USA). The tissue protein content was quantitatively analyzed using the BCA protein Assay kit (Pierce). Equal amounts of proteins (30 ug) were separated using 10% SDS polyacrylamide gels and then transferred to a PDVF membrane. The PDVF membrane was blocked with 5% skim milk diluted with Tris-buffered saline containing Tween 20 (TBS-T) for 90 min and the incubated with primary anti-KCNK2 antibodies (1:200 dilution, Sabin), KCNK9 (1:1000 dilution, Abcam), KCNK15 antibodies (1:1000 dilution, Absin), or KCNK17 antibodies (1:800 dilution, Absin) at 4 °C overnight followed by incubation with secondary goat anti-rabbit IgG (1:1000 dilution, abclonal) at 37 °C for 1 h. Immunodetection was performed through chemiluminescence (ECL, Guangzhou, China), and β-actin (1:1000 dilution, abclonal) or GAPDH (1:2000 dilution, abclonal) was used as loading control.

The animal total protein extraction kit (Bestbio, China) and RIPA lysis buffer (Solarbio, China) were used for protein extraction of PSCs and LX-2 cells, respectively. After quantifying protein concentration using BCA kit (Bestbio, China), 20 μg of protein samples were separated in 12% SDS-PAGE gel and transferred to nitrocellulose membranes (Merck, Germany). The membrane was sealed with 5% skimmed milk at room temperature for 2 h, and then the primary antibodies, CE positive/negative sheep sera, polyclonal antibodies (anti-rEgE2D2 and rEgE2N), pre-immunized rat sera (1:200 v/v dilution), α-SMA, COL1A1, and GAPDH (1:5000 v/v dilution, ABclonal, China) were incubated overnight at 4 °C. After fully washing the membrane, corresponding horseradish peroxidase (HRP)-labeled secondary antibodies (goat anti-rat IgG, goat anti-rabbit IgG, and rabbit anti-sheep IgG (1:2000 v/v dilution, ABclonal, China) were added and incubated at room temperature for 1 h. Metal Enhanced DAB Substrate Kit (20 ×) (Solarbio, China) was used to visualize the bands.

Total protein of tissues was extracted through the ice-cold radioimmunoprecipitation lysis buffer (RIPA, Thermo Fisher Scientific, 89,900), which contained the protease inhibitor cocktail (Merk, P8340). Subsequently, the extracted proteins were quantified through the BCA Assay Kit (Beyotime, P0010) and boiled for degeneration. Then, we separated the proteins in SDS-PAGE (Yeasen, 20315ES05) and transferred them into the PVDF membrane (Merk, 3,010,040,001). After being blocked into 5% Bovine serum albumin (BSA, Yeasen, 36104ES25), the PVDF membrane was then incubated with primary antibodies: Anti-beta-actin (Proteintech, 20,536, 1:1000), Anti- ZBTB16 antibody (ABclonal, A5863, 1:1000), Anti-SERPINA10 antibody (ABclonal, A7106, 1:1000), and Anti-CD38 antibody (ABclonal, A1680, 1:1000). Stepwise, the membranes were incubated in secondary antibodies: Goat Anti-Mouse IgG (Proteintech, SA00001-1, 1:1000) and Goat Anti-Rabbit IgG (ABclonal, AS014, 1:1000), followed by enhanced chemiluminescence to display bands.

The renal tissues were lysed in sodium dodecyl sulfate (SDS) lysis buffer (Beyotime, China) for extracting total protein, and the concentration of protein was determined using the bicinchoninic acid protein assay kit (Thermo Fisher, USA). Then, 40 µg total protein was loaded into each lane and separated by SDS-polyacrylamide electrophoresis. The proteins in the SDS gel were transferred onto a polyvinylidene difluoride membrane (Millipore, USA), and the nonspecific binding protein was blocked in Tris-buffered saline with Tween 20 (TBST) buffer containing 5% skimmed milk for 1 h at 37°C. The membrane was incubated using primary antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:5000; Abcam), FADD (1:100; Abcam), Apaf-1 (1:2000; Abcam), and CHOP (1:1000; Abcam) overnight at 4°C on a shaking table. Sequentially, the membrane was immersed in goat anti-rat immunoglobulin G (IgG; 1:5000; ABclonal, USA) or goat anti-rabbit IgG (1:5000; ABclonal) antibody for 1 h at room temperature. The membrane was washed three times using TBST for 10 min after each step. The protein bands were visualized using an enhanced chemiluminescence system detection kit (ECL; Amersham, USA) in a bioimage system (Bio-Rad, USA). The grayscale value was calculated using Image-Pro Plus 6.0 software. Five samples were selected randomly for western blot analysis.

Total protein from fracture tissues was extracted from all groups six weeks after the operation, followed by measuring concentration of protein extract with a BCA kit (Beyotime, Shanghai). Equal amounts of protein samples were subjected to sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) electrophoresis and transferred to polyvinylidene fluoride (PVDF, Millipore, MA) membrane. The obtained PVDF membrane was incubated overnight with primary antibodies against USP1 (1:3000, Proteintech, Wuhan), BMP2 (1:1000, Affinity, Changzhou), RUNX2 (1:1000, Abclonal, Wuhan), OCN (1:500, Abclonal, Wuhan), Akt (1:1000, Affinity, Changzhou), p-Akt^T308 (1:1000, Affinity, Changzhou) and p-Akt^S473 (1:1000, Affinity, Changzhou) at 4 °C. At last, after washed by TBST (Tris-buffered saline + Tween), the membranes were incubated with secondary antibodies goat anti-rabbit IgG (Abclonal, Changzhou) and goat anti-mouse IgG (Abclonal, Wuhan) at 37 °C for 40 min. The bands were detected by enhanced chemiluminescence (ECL, Beyotime, Shanghai) detection reagent and the optical density values of the target bands were analyzed by image analysis software (Tanon, Shanghai).

Fetal bovine serum (FBS) was obtained from Natocor (Cordoba, Argentina). High glucose Dulbecco's modified Eagle's medium (DMEM) was purchased from HyClone (Logan, Utah, USA). Penicillin, streptomycin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), phosphate buffer saline (PBS), LPS, and sodium dodecyl sulfate (SDS) were obtained from Sangon Biotech (Shanghai, China). Enhanced BCA Protein Assay Kit and 4,6-diamidino-2phenylindole (DAPI) were obtained from Beyotime (Shanghai, China). Griess B (1%(w/v) sulphanilamide containing 5% (w/v) H₃PO₄, Griess A (0.1% (w/v) N-(1-naphthyl)-ethylenediamine dihydrochloride), RIPA buffer, dimethyl sulfoxide (DMSO), and sodium nitrite were provided by Solarbio (Beijing, China). ELASA kits of TNF-α and PGE2 were obtained from Boster (Wuhan, China). The primary antibodies for iNOS, COX-2, p38, p-ERK, p-p38, ERK, JNK, p-JNK, β-actin, NF-κB p65, c-Fos, p-Akt, c-Jun, PDK1, GAPDH, p-PDK1, p-IκBα, Akt, and IκBα, and the secondary antibody goat antirabbit (IgG) were provided by AB Clonal Biotech (Wuhan, China). Other standards and analytical grade reagents were obtained from Tianjin ZhiYuan Reagent Co., Ltd. (Tianjin, China).