Mouse anti cd4

Endobronchial biopsies were performed using disposable biopsy forceps with jaws closed at the interbronchial carina before retracting. Tissue was fixed immediately in 4% paraformaldehyde and paraffin embedded. CD4⁺ T cells were identified by staining with mouse anti-CD4 (Dako) at 1:100 dilution using the EnVision peroxidase staining method (Dako) as previously described (62 (link)). Slides were coded to avoid observer bias and assessed using a Leitz Dialux 20 light microscope and Image 1.5 software. Total epithelial and subepithelial areas of 2 to 3 bronchial biopsies were counted at each time point. Cell counts were expressed as the number of cut cell profiles with visible nucleus per mm² of subepithelium and per 0.1 mm² of epithelium. The coefficient of variation for repeat counts of positive cells by a single observer ranged from 5% to 6%.

Fresh tissue and cultured cells were fixed in 4% paraformaldehyde, and immunostaining was performed using primary antibodies prepared in different species: rabbit anti-nestin (1:200, Chemicon International, Temecula, CA, USA), rabbit anti-Isl-1 (1:200, Chemicon International), mouse anti-Vimentin (1:500, Chemicon International), rabbit anti-PDX-1(1:500, Chemicon International), mouse anti-glucagon (1:1000, Sigma-Aldrich), guinea pig anti-insulin (1:100, Zymed Laboratories, South San Francisco, CA, USA), mouse anti-C-peptide (1:250, Chemicon International), rabbit anti-CD3 (ready to use, ZSGB-BIO, Beijing, China), mouse anti-CD4 (1:60, Dako, Glostrup, Denmark), mouse anti-CD8 (1:80, DAKO) and mouse anti-CD68 (1:80, DAKO). Binding of the primary antibody was visualized by either the immunofluorescence technique or an HRP enzymatic reaction using 3,3′-diaminobenzidine (DAB) as the chromogen. For Prussian blue staining, fixed cells and tissue sections were incubated in a 1:1 mixture of 4% potassium ferrocyanide and 4% HCl for 50 minutes. DAPI or nuclear fast red were used as nuclear counterstains. Representative images were captured using an Olympus BX53 Microscope or NIKON2000U microscope.