Hrp conjugated secondary antibody

RAW264.7 cells were harvested at 24 h and 48 h post-infection and lysed in lysis buffer on ice for 40 min. The supernatant was obtained by centrifugation at 13,000 rpm for 20 min at 4 °C. Protein concentration was determined using the bicinchoninic acid assay. Total cellular protein was extracted by boiling lysates for 10 min in 5× sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer. Proteins were separated by SDS-PAGE on a 12% polyacrylamide gel and then transferred onto polyvinylidene fluoride membranes. The membranes were blocked for 2 h at room temperature in Tris-buffered saline containing 0.5% Tween-20 (TBST) and 10% skimmed milk and then incubated overnight at 4 °C in blocking solution containing antibodies against Bax (1:2000; Abmart, Shanghai, China), Bcl2 (1:2000; Abmart), cytochrome C (1:5000; Abcam, Cambridge, MA, USA), Caspase-9 (1:1000; Proteintech, Wuhan, China), Caspase-3 (1:1000; Proteintech), or β-actin (1:1000; Proteintech). The membranes were washed four times with TBST for 8 min, incubated for 2 h with the corresponding HRP-conjugated secondary antibody (1:5000; Zhongshan Golden Bridge Biotechnology, Beijing, China), and washed four times in TBST for 8 min. The signal was visualized using an ECL chemiluminescence kit (Beyotime, P0018FS, Shanghai, China) and imaged by the Gel Image System (Tannon Biotech, Shanghai, China).

Placenta samples were fixed in 4% paraformaldehyde (Sigma, MO, USA) and embedded in paraffin wax. The tissue was then cut into to 5μm sections. The sections were subjected to deparaffinization, rehydration and antigen recovery before being incubated with antibodies against human SGPL1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C. The sections were then incubated with HRP-conjugated secondary antibody (Zhongshan Goldenbridge Biotechnology Co., Beijing, China) for 30 min at room temperature and visualized with DAB.

Four micron-thick tissue sections were deparaffinized in xylene, rehydrated, subjected to microwave antigen retrieval in citrate buffer (10mM, PH 6.0) for 20 min, and then cooled at room temperature. Endogenous peroxidase activity was quenched by soaking in 3% hydrogen peroxide. Sections were then blocked with normal goat serum at room temperature to prevent non-specific binding. Subsequently, individual sections were incubated overnight at 4°C with rabbit anti human VEGFR-2 polyclonal antibody (Zhongshan Golden Bridge Biotechnology Inc), goat anti human CCR1 polyclonal antibody or rabbit anti human EpCAM polyclonal antibody (Abcam). After rinsing in phosphate-buffered saline (PBS), the sections were incubated for 30 min with HRP-conjugated secondary antibody (Zhongshan Golden Bridge Biotechnology Inc). After subsequent washing, immunoreactivity was visualized with the chromogen 3,3′-diaminobenzidine. Finally, all slides were counterstained with hematoxylin, dehydrated, and mounted.

Western blot analysis was performed as previously described [46 (link)]. Cells were collected and lysed in RIPA buffer (Solarbio, China) plus phenylmethysulfonyl fluoride (PMSF) on ice for 30 min, followed by centrifugation at 12000 g for 20 min. The protein concentration in the extract was measured with a BCA Protein Assay Kit (Pierce, USA). After boiling for 10 min to denature the protein, the samples were diluted in 4× loading buffer (Beyotime, China) and loaded onto SDS-PAGE (10%) gels for electrophoresis and transferred to PVDF membranes (Millipore, USA). BenchMark Pre-Stained Protein Standard (Invitrogen, USA) was used as the size marker. The membranes were blocked with TBST containing 5% non-fat milk for 1 hour, followed by immunoblotting with primary antibodies at 4°C overnight. After incubation with HRP-conjugated secondary antibody (Zhongshan Goldenbridge Biotechnology Company, China) for 1 h, the protein bands were evaluated using enhanced chemiluminescence (Millipore, USA) and detected with a LAS-4000 MINI System (GE, USA). β-Actin or β-tubulin was used as an endogenous control. All the experiments were repeated three times. Antibodies for VRK1 (1:100000), c-Jun (1:1000), phospho-c-Jun (Ser63) (1:5000), c-MYC (1:10000), β-actin (1:1000) and β-tubulin (1:1000) were purchased from Abcam.

To validate the accuracy of MS quantification for PGRMC1, PGRMC1 concentration in tissue or serum sample was also detected by Western blotting. 40ug tissue proteins, or 20μg serum samples without high-abundant proteins were separated on a 12% SDS-PAGE gel, then transferred onto a PVDF membrane. Subsequently the membrane was incubated in TBST buffer (20mM Tris-HCl, pH 7.6, 150mM NaCl, 0.1% Tween-20) with 5% non-fat milk to block nonspecific binding at room temperature for 1 h. Then the PVDF membrane was incubated with the anti-PGRMC1 antibody (ab48012, Abcam) at a dilution of 1:1000 in TBST with 1% non-fat dry milk overnight at 4°C. The PVDF membrane was washed with the blocking solution and incubated with the HRP-conjugated secondary antibody with a dilution of 1:10000 (ZhongShan-Golden Bridge, China) at 37°C for 1h. Detection was carried out using the ECL reagent (Amersham Biosciences, Piscataway, New Jersey, USA). For the cellular protein sample, β-actin was taken as a comparison control for western blotting.

Brain sections were cut from paraffin-embedded brain tissue blocks. Brain sections were dewaxed, hydrated, and treated for antigen retrieval in citrate buffer at 100 °C for 20 min. Paraffin-embedded tissue sections were used for H&E staining. For IHC, tissue sections were first incubated with goat serum (Zhongshan Golden Bridge Bio-technology, Beijing, China) for 30 min at room temperature, then incubated with the primary antibody Ki-67 (9109, CST, 1: 200) and CD31 (ab28364, abcam, 1:50) at 4 °C for 12 h and subsequently incubated with HRP-conjugated secondary antibody (Zhongshan Golden Bridge Bio-technology, Beijing, China) for 1 h at room temperature. Sections were incubated with diaminobenzidine (DAB) (Zhongshan Golden Bridge Bio-technology, Beijing, China) followed by image acquisition with a microscope.