Mouse anti human cd24 antibody

Double immunofluorescence staining was performed to examine the co-expression patterns of ANXA10 with CD24 (CSC marker for PDAC) in PanIN, IPMN, PDAC, and normal tissues, respectively. The slides were deparaffinized, rehydrated, and treated with citrate buffer for antigen retrieval and then 2% BSA to block non-specific binding as described above. To achieve double IF staining of ANXA10 and CD24, rabbit anti-human ANXA10 antibody (1:250, Abcam, Cambridge, MA) was mixed with mouse anti-human CD24 antibody (1:150, Abcam, Cambridge, MA). The antibody mixture was then incubated with the slides overnight at 4°C. Subsequently, DyLight 488 anti-rabbit IgG (green) and DyLight 549 anti-mouse IgG (red) (Vector laboratories, Burlingame, CA) were diluted (1:150) and incubated with the slides for 1 hr at room temperature. Nuclei visualization was explored by DAPI counterstaining (blue).

Mouse Anti-human CD24 antibody (Cat.: ab30350), rabbit anti-human CD44 antibody (Cat.: ab157107), rabbit anti-human CD133 antibody (Cat.: ab19898), rabbit anti-human Oct4 antibody (Cat.: ab19857), rabbit anti-human Nanog antibody (Cat.: ab21624), rabbit anti-IRE1 (phosphor S724) antibody (Cat.: EPR5253), rabbit anti-IRE1 antibody (Cat.: ab96481), rabbit anti-PERK (phosphor T982) antibody (Cat.: ab192591) and rabbit anti-PERK antibody (Cat.: ab65142) were obtained from Abcam (Cambridge, England) and diluted followed the manufacturer’s instructions.
Total protein was prepared using RIPA buffer (Thermo Scientific, Waltham, MA, USA) followed the manufacturer’s instruction. The same amount of protein from total protein was fractionated using Tris-glycine gels, and transferred to PVDF membranes. After transferring, PVDF membranes were blocked in 5% milk/TBS buffer at room temperature for 30 min, and this was followed by an incubation with primary antibodies at 4°C overnight. After washing with PBS-T (containing 0.1% Tween-20) for three times, HRP-conjugated secondary antibodies were incubated with PVDF membranes for another 1 hour. Blots were developed using Pierce^™ ECL Western Blotting Substrate (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions.

Protein expression was determined by immunofluoresence (IF) staining [7 (link)]. Briefly, the tissue specimens were placed in xylene twice for 20 minutes and hydrated in 100% ethanol, 95% ethanol, 75% ethanol, 5 minutes each and water for 3 minutes. Antigen retrieval was completed by boiling the sections in solution (BioGenex HK080-9K) for 15 minutes. Subsequently, these were blocked with 2% BSA in PBS containing 0.05% Tween (PBST) for 1 hour at room temperature and incubated with rabbit anti-human CD90 antibody (1:100, Abcam, Cambridge, MA) or mouse anti-human CD24 antibody (1:150, Abcam, Cambridge, MA) at 4°C overnight. The secondary antibodies of DyLight 488 anti-rabbit IgG (green) or DyLight 549 anti-mouse IgG (red, Vector Laboratories, Burlingame, CA) were used at 1:150 dilution and incubated in a dark place for 1 hour at room temperature. CD90 staining was visualized in green and CD24 in red. Staining with DAPI (blue, 1:4000 dilution) presented nuclei visualization. The sections were dehydrated in alcohol and xylene, then cover-slipped with TM mounting medium (Sigma, USA).