CD19 CD79A PDGFRB brain and tonsil staining in humans

Human tonsil (CD79A and CD19 positive controls) and brain (test tissue) were stained for CD19, CD79A, PDGFRB to examine whether CD19 appeared to be expressed in brain mural cells. Protocol: Samples were blocked for 2hrs at room temperature in blocking buffer (PBS with 1mM CaCl2, 1mM MgCl2, 20% Donkey serum (Jackson Immuno Research: 017-000-121), 0.3% Triton X-100). Samples were stained in staining buffer (PBS with 1mM CaCl2, 1mM MgCl2, 1% Donkey serum, 0.3% Triton X-100). Antibody dilutions were as follows: goat anti-PDGFRβ 1:100 (Novus Biologicals: AF385), rabbit anti-CD79a 1:100 (Fisher Scientific PIMA516344; clone SP18), mouse anti-CD19 1:50 (GeneTex: GTX42325, clone LE-CD19). Note that CD19 primary (1:50) and secondary (1:50) were used at higher concentrations than other channels to address any concerns over lack of sensitivity. Our positive control in human tonsil showed abundant signal as expected indicating the efficacy of our staining procedure. Slides were washed 3x with PBS (5 min each), and stained in staining buffer with secondary antibodies, each from Jackson Immuno Research: 1:50 Alexa Fluor 488 AffiniPure Donkey Anti-Mouse IgG (H+L) (715-545-151); 1:100 Rhodamine Red-X (RRX) AffiniPure Donkey Anti-Rabbit IgG (H+L) (711-295-152); 1:100 Alexa Fluor 647 AffiniPure Donkey Anti-Goat IgG (H+L) (705-605-147). Slides were then washed 3x with PBS (5 min each) and mounted in Prolong Diamond Antifade with DAPI (Fisher Scientific: P36962) and sealed with nail polish. Images were all collected using the same laser settings on a Leica SP8 confocal microscope.