Mouse anti human thrombin antibody

To determine basal prothrombin protein localization in fetal membranes, freshly harvested, full-thickness fetal membranes without U. parvum infection were fixed in 10% formalin for IHC. Paraffin sections were de-waxed and rehydrated. After heat-induced antigen retrieval, the sections were stained using the anti-mouse HRP-DAB Cell & Tissue Staining Kit following manufacturer’s instruction (R&D Systems). Mouse anti-human thrombin antibody (Abcam) was used at a 1:500 dilution in PBS with 1% BSA and 5% goat serum. Placenta tissue was used as positive control. For each batch of staining, one slide was incubated with normal mouse IgG (Abcam) instead of the primary antibody as a negative control. Images were taken using a Zeiss Axio Observer (20X). Three rounds of IHC staining were performed using fetal membrane tissues collected from three subjects.

Cell lysates were obtained using radioimmunoprecipitation assay (RIPA) buffer (Sigma) with the complete mini-protease inhibitor cocktail (Roche). For frozen tissue explants and placenta (positive control for Western blots), tissues (~500 mg) were pulverized then homogenized in 1.0 ml of RIPA buffer containing protease inhibitor. Purified plasma prothrombin (Enzyme Research Laboratories) was used as the positive control. Western blot analysis was performed using a standard procedure. Rabbit anti-human GAPDH antibody (1:20,000, Cell Signaling Technology), mouse anti-human thrombin antibody (1:2000, Abcam), and the secondary antibodies (1:2500, Cell Signaling Technology) were used. The primary human thrombin antibody detects both prothrombin and thrombin. Film development was optimized to maintain bands within the linear range. Band intensity was measured using ImageJ analysis software (National Institute of Health [NIH], Bethesda, MD). The densitometry of the protein bands were normalized to GAPDH and then compared and presented as ratios. Five replicates were performed using fetal membrane tissues or primary fetal membrane cells collected from five subjects.