Alexa fluor 647 anti mouse cd19 antibody

The extraorbital and intraorbital glands were extracted and fixed in optimal cutting temperature compound (Sakura Finetek USA, Inc., Torrance, CA). The glands were preserved at -80°C until sectioning. The sections were cut at 4 μm and subjected to hematoxylin-eosin staining and immunostaining for GFP, CD3, CD11b, and CD19. The primary antibodies used were Alexa Fluor^® 488 anti-GFP antibody, anti-mouse Alexa Fluor^® 700 CD3 antibody, Alexa Fluor^® 594 anti-mouse CD11b antibody, and Alexa Fluor^® 647 anti-mouse CD19 antibody (all from BioLegend^®, San Diego, CA). A mounting medium containing DAPI (VECTASHIELD^® Mounting Medium, Vector Laboratories, Inc., Burlingame, CA) was used for counterstaining.
For TUNEL staining, ApopTag^® Red in situ Apoptosis Detection Kit (EMD Millipore, Billerica, MA) was used.
To detect MSCs in the glands, the sections were stained with mouse anti-human nuclei (Cy3 conjugate, 1:50) (MAB1281C3, Millipore, Billerica, MA) and mouse anti-human mitochondria (1:50) (MAB1273, Millipore), followed by Alexa Fluor 488 goat anti-mouse IgG (1:500) (A11001, Molecular Probes^®/Life Technologies, Eugene, OR).

Paraffin-embedded tissue sections at thickness of 8 μm were used in this study. Coronal sections were prepared from 1 mm behind the bregma. For immunostaining, the following primary antibodies were used: anti-CD4, C-Terminal antibody produced in rabbit (1: 200, SAB4503583, Sigma-Aldrich), Purified Rat Anti- Mouse CD8a (1: 200, 550281, BD Biosciences), Alexa Fluor® 647 anti-mouse CD19 Antibody (1: 100, 550281, BioLegend), Purified Rat Anti-Mouse Ly-6G (1: 200, 550291, BD Biosciences), Alexa Fluor® 488 anti-mouse CD45.2 Antibody (1: 100, 109815, BioLegend). Primary antibodies were incubated at 4°C overnight, followed by incubation with species-specific Alexa Fluor (488 and 594)-conjugated secondary antibodies for 1 h. Pictures were acquired with a Nikon Eclipse T300 fluorescence microscope and analyzed using Image Pro Plus (Media Cybernetics, Inc. Rockville, MD). For cell counting, positive cell numbers were counted in every tenth tissue section through the entire tissue block (25 (link)–27 (link)).