Anti human cd45

Lungs and kidneys from control and pristane-injected hu-mice and NSG were harvested at eight weeks post-injection, fixed with 10% formalin and embedded in paraffin for processing into 5 µm tissue sections. Rehydrated lung and kidney sections were stained with Hematoxylin & Eosin (H&E) (Thermo Scientific), and evaluated by a pathologist who was blinded of the samples’ identity. Glomerular enlargement was quantified by area measurement of 50 random glomeruli from kidneys of each experimental animal. Images were captured using brightfield slide scanner (Axio Scan Z1) and processed by Zen software (Carl Zeiss). For immunohistochemistry (IHC), kidney sections were subjected to heat-mediated antigen retrieval with sodium citrate (pH6) buffer prior to staining with appropriate antibodies. IHC staining was performed using the SuperPicture 3^rd Gen IHC Detection Kit (Life Technologies) according to manufacturer’s instruction. Primary antibodies used in the study include anti-human IgG, anti-human IgM (Bethyl Laboratories), and anti-human CD45 (AbCAM). Anti-mouse, anti-rabbit and anti-goat HRP conjugated secondary antibodies were purchased from Life Technologies.

Multiple organs including brain, heart, lungs, liver, kidneys, forelimbs/hind limbs, and spleen were removed from sacrificed mice at endpoint. The organs were fixed in 10% formalin, embedded in paraffin wax, processed to obtain 5 μm sections, and subjected to Hematoxylin & Eosin (H&E) (Thermo Fisher Scientific) or immunohistochemistry staining following established protocols. Primary antibodies including anti-human CD45 (cat# ab781), anti-human MPO (cat# ab134132), and anti-human c-kit (cat# ab32363) monoclonal antibodies were purchased from Abcam and used for immunohistochemistry. The primary antibody was detected using Rabbit specific IHC polymer detection kit HRP/DAB (AbCam) or Mouse on Mouse Polymer IHC Kit (AbCam) following manufacturer’s instructions. Histopathological images were acquired using Axio Scan. Z1 slide scanner (Zeiss) and analyzed using Zen 2 (blue edition; Zeiss) software.

HBMSCs were isolated from bone marrow that was collected from the iliac crest of 4 healthy volunteers whom given informed written consent. The hBMSCs derived from each volunteer were isolated as previously described 26 (link). Phenotypic identification of cultured hBMSCs was performed by standard flow cytometry. hBMSCs (passage 3-7, 1×10⁶ cells) were incubated for 60 min with PE- or FITC-conjugated mouse monoclonal antibodies, namely, anti-human CD29, anti-human CD45 (Abcam, Cambridge, UK), anti-human CD34 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), or anti-human CD90 (BD Biosciences, San Jose, CA, USA), or with an immunoglobulin isotype as a negative control. The cells were analyzed with a FACS Calibur system (FC500, Beckman Coulter, Fullerton, CA, USA) after washing. PHHs were isolated using a four-step collagenase perfusion method as previously described 27 (link). Briefly, the liver segments were perfused by the extracorporeal circulating perfusion apparatus with four different buffer solutions supplemented with 0.37 mg/ml EDTA, 0.5% dispase, 0.05% collagenase type IV (Gibco BRL, Grand Island, NY), and 40 µg/ml DNase I (Sigma, St. Louis, MO). The remnants were dispersed mechanically with a scalpel blade and filtered sequentially through a 100 µm nylon mesh. The isolated cells were harvested and centrifuged at 4 °C and 50 g for 3 min.