Grp78 antibody

Staining of tissue sections was performed as previously described [30 (link)]. Briefly, slides were deparaffinized in xylene and rehydrated in graded alcohol. Antigen retrieval was performed using microwave antigen retrieval method with Target Retrieval Solution, pH 6.10. Endogenous peroxidase activity was quenched by 0.3% H₂O₂ in water. After washing the slides to reduce non-specific binding, they were incubated with specific antibodies to thrombospondin-1 (Clone A6.1 Santa Cruz Biotechnology). Staining was done using DAB as the chromogen. mRNA expression of CD47 was assessed by real-time PCR using specific primers (Forward Primer 5′-AGCATGGAATGACGACAGTG-3′, reverse primer 5′-GATGTGGCCCCTGGTAGC-3′). Cell surface expression of CD47 was measured using a BD LSRFortessa X-20 Analyzer Flow Cytometer, staining of CD47 was performed using FITC Human Antibody B6H12 (Biolegend). Confirmation of GRP78 knockdown was performed by Western blot hybridization using a GRP78 antibody from Cell Signaling Technologies.

The tissues were embedded in paraffin wax after cutting and dehydrating them with serial alcohol solutions. The paraffin-embedded tissues were cut into 3-μm sections and placed on slides, followed by staining with the GRP78 antibody (1:1000; #3183; Cell Signaling), ki-67 (1:500; SP6; Spring Bioscience, Pleasanton, CA, USA), and α-SMA (1:100; SP171; Spring Bioscience, Pleasanton, CA, USA) for immunohistochemical (IHC) analysis according to the manufacturer’s instructions. For H&E staining, the paraffin embed slides were dewaxed, gradually hydrated through graded alcohol, and were stained in hematoxylin solution and differentiated in 1% hydrochloric alcohol. After rinsing with distilled water, the sides were dehydrated in 95% ethanol, counterstained in 1% eosin solution, washed with 70% ethanol, absolute ethanol, and then were cleared in 2 changes of xylene.