Cd3 sp7

6 micron sections of synovium frozen in OCT were fixed in acetone, rehydrated in PBS, and blocked with 10% normal goat serum prior to application of primary antibodies as follows: PD-1 (EH12.2H7, BioLegend), CD3 (SP7, Abcam), CD20 (L26, Dako), CXCR5 (MAB190, R&D Systems), all at a dilution of 1:100 except for CD20, which was used at 1:300. All secondary antibodies were raised in goat. CXCR5 was detected using anti mouse IgG2b biotin (Southern biotech) followed by streptavidin conjugated AlexaFluor 546 (Life Technologies), CD20 with anti-mouse IgG2a FITC (both Southern Biotech), PD-1 with anti-mouse IgG1 conjugated to AlexaFluor 647 and CD3 with anti-rabbit AlexaFluor 546 (both Life Technologies). FITC staining was amplified with anti-FITC AlexaFluor 488 (Life Technologies). Slides were mounted using ProLong Diamond (Life Technologies), left to cure overnight and imaged using a Zeiss LSM 780 confocal microscope. Images were processed using Zen Black (Zeiss) and then ImageJ. Cell counts were performed on images obtained from confocal imaging using the Cell Counter plugin for ImageJ (imagej.net/Cell_Counter). Synovial regions were categorized as ‘lymphoid aggregates’ when the B cells and T cells formed distinct clusters, and ‘diffusely infiltrated’ when B cells were loosely distributed within the synovium.

For histological analysis, pancreatic specimens were fixed with 10 % buffered formalin, dehydrated in ethanol, embedded with paraffin, and stained with H&E or Gomori Trichrome. The percentage of preserved acinar area and fibrosis were calculated, as previously described (Zambirinis et al., 2015 (link)). Immunohistochemistry on paraffin-embedded or frozen mouse tissues was performed using antibodies directed against F4/80 (CI:A3-1), Arginase1 (Polyclonal), CD3 (SP7; all Abcam, Cambridge, MA), RIP1 (#5389; ProSci, Poway, CA), CK19 (Troma-III; University of Iowa), and DAPI (#H-1200; Vector Labs, Burlingame, CA). For paraffin-embedded slides, samples were dewaxed in ethanol followed by antigen retrieval with 0.01 M Sodium Citrate with 0.05 % Tween. For frozen slides, samples underwent antigen retrieval (10% SDS) prior to incubation with the primary antibody. Immunofluorescent images were acquired using a Zeiss LSM700 confocal microscope with ZEN 2010 software (Carl Zeiss, Thornwood, New York).