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3 protocols using rabbit anti fap

1

Comprehensive Tumor Microenvironment Analysis

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Harvested tumors were embedded in O.C.T. medium (TissueTek, Torrance, CA). 4-5 μm frozen sections were fixed in ice-cold acetone for 20 minutes, blocked with 1% bovine serum albumin in phosphate buffered saline, and immunostained using standard protocols. Primary antibodies used are: goat anti-Collagen 1 (Southern Biotech) 1:200, mouse anti-αSMA-FITC/mouse anti-αSMA-Cy3 (Sigma) 1:200, rat anti-mouse Ki67 (abcam) 1:200, rabbit anti-FAP (abcam) 1:200; rat anti-foxp3 (eBioscience) 1:150; rabbit anti-NG2 (Millipore) 1:200; rat anti-CD31 (BD Pharmingen) 1:200, mouse 4.3.11.3 (hypoxyprobe) 1:50, and rabbit anti-LOX (Imgenex) 1:200. Stainings for pimonidazole adduct (hypoxyprobe) was processed using the M.O.M. Kit (Vector Labs) according to the manufacturers' recommendations. Apoptosis was assessed using in Situ Cell Death Detection Kit, TMR red (Roche). DAPI was used to stain cell nuclei.
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2

Immunohistochemical Analysis of Pancreatic Tissue

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Paraffin sections of pancreata from ND adults, newborns and T2D donors were obtained from the Network for Pancreatic Organ donors with Diabetes (nPOD). Antigen retrieval was performed by pressure cooker heating (Prestige Medical, Northridge, CA) using citrate buffer (pH 6.0). Sections were blocked with CAS-Block (Invitrogen). Primary antibodies used in this study included guinea pig anti-insulin (1:500; DAKO), rat anti-CD44 (1:100; Invitrogen), rabbit anti-vimentin (1:100; Abcam), rabbit anti-FAP (1:100, Abcam), mouse anti-glucagon (1:400, Cell Signalling). For DNA counterstain, we used 4′,6-diamidino-2-phenylindole (DAPI; 1:100; Sigma–Aldrich). Secondary antibodies were all from Jackson Immunoresearch Laboratories (1:250; West Grove, PA, USA). For double staining, we used only affinity-purified secondary antibodies suitable for multiple labelling. All immunofluorescence images were captured on Olympus FV1000 confocal microscope.
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3

Western Blotting Procedure for Protein Analysis

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Western blotting was performed as described previously (47 (link)). A total of 5 μg protein was mixed with 4× Laemmli sample buffer (Bio-Rad Laboratories) and boiled at 95°C for 5 minutes. Each protein sample was separated by SDS-PAGE in 4%–20% TGX-GEL (Bio-Rad Laboratories) and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories) by using a semidry method. The membranes were blocked in 5% skim milk in Tris-buffered saline (TBS) containing 0.05% Tween 20 (TBS-T) for 1 hour with shaking at RT. Afterward, the membrane was incubated overnight with shaking at 4°C with primary antibodies, which cross both human and mice — rabbit anti–E-cadherin (1:1000, 24E10, Cell Signaling Technology), rabbit anti-vimentin (1:1000, Abcam), or rabbit anti-FAP (1:500, Abcam) — and then incubated with horseradish peroxidase–conjugated (HRP-conjugated) secondary antibodies for 1 hour with shaking at RT. Washes before and after antibody reactions were done on a shaker, 3 times within TBS-T for 10 minutes each at RT. Alternatively, before the detection of β-actin, we performed the stripping procedure for 10 minutes because the molecular weight of vimentin and β-actin are similar. Then, the membranes were incubated with HRP-conjugated mouse anti–β-actin (1:20,000, Abcam) antibody for 1 hour with shaking at RT. Blots were visualized with ECL substrate (Bio-Rad Laboratories).
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