Ab48012

The PGRMC1 concentration in human serum was measured by the ELISA (CUSABIO, CSB-EL017876HU). Serum samples were added into a 96-well plate with pre-coated anti-PGRMC1 antibody (ab48012, Abcam) to incubate for 1h at 37°C, then the plate was washed with PBST buffer (3.2 mM Na₂HPO₄, 0.5 mM KH₂PO₄, 1.3 mM KCl, 135 mM NaCl, 0.05% Tween 20, pH 7.4) for 5 times, and incubated with the secondary antibodies for 1h at 37°C. Being stopped by adding 1 N HCl, the absorbance in the 96-well plate was detected at 450 nm on an ELISA Reader (Multiskan Mk3, thermo) with the correction factor at 570 nm.

Tissue samples were paraffin-embedded to cut into sections with 4μm thickness for hematoxylin-eosin (HE) and immunohistochemistry (IHC) analysis mainly according to our previous protocols [21 (link)]. The primary anti-PGRMC1 antibody (ab48012, Abcam) was used at a dilution of 1:200. The second antibody was a biotinylated anti-goat IgG (ZB-2306, ZSGB-BIO Corp., Beijing, China). Tissue slices were visualized by 3, 3-diaminobenzidine solution and counterstained with hematoxylin. Meanwhile the primary antibody was substituted by phosphate-buffered saline (PBS) serving as a control sample.
An estimated percentage of IHC staining was determined by calculating the average number of positive stained cells from 3–4 microscopic fields under 400-fold magnification. The score for positive staining cells was defined as 0–4 with the percent of positive cells separately ranged from 0–5%, 6–25%, 26–50%, 51–75% and over 75%. Similarly the staining intensity was divided into 4 levels: 0, negative; 1, weak; 2, moderate; 3, strong. The final immunoreactivity score for each tissue slice was defined as the staining intensity multiplied by percentage of positive cells [22 (link)]. The scores, ranging from 0 to 12, indicate different PGRMC1 abundance in tissues, including a negative (0, -); weak (1–3, +), moderate (4–7, ++) and strong PGRMC1 abundance (8–12, +++).

The in-situ proximity ligation assay (PLA) procedure was performed with the Duolink® PLA Kit (Sigma-Aldrich, DUO92008) and following the manufacturers protocol. The cells were incubated with the primary antibodies i.e., anti-PGRMC1 (Abcam, ab48012) with PHB1 (Abcam, ab75766) and PHB2 (Cell signaling, 14085S) overnight at 4 °C. The slides were washed twice for 5 min with buffer A, followed by incubation with the PLA probes (anti-goat PLUS and anti-rabbit MINUS) in antibody diluent for 60 min at 37 °C. After washing twice for 5 min with buffer A, ligation was performed using ligase diluted in ligation buffer for 30 min at 37 °C. Then the cells were washed with buffer A before incubation for 100 min with amplification stock solution at 37 °C. After washing twice for 10 min with buffer B, nuclear DNA was labeled with DAPI for 10 min and slides were mounted with mounting medium. Negative PLA control was performed using respective isotype control antibodies (isotype goat, Abcam, ab37373; isotype rabbit, Abcam, ab37415). Red fluorescence dots inside the cellular areas representing a single protein–protein interaction were quantified using image J software.