Anti calbindin d 28k antibody

Mouse brains were collected and fixed in 4% paraformaldehyde solution in PBS (Thermo Fisher Scientific, J19943K2) for 7 days at 4 °C. Tissues were transferred to 10% sucrose in PBS for 1 day at 4 °C, followed by 20% sucrose in PBS for 1 day at 4 °C and finally 30% sucrose in PBS for 1 day at 4 °C. The brain samples were processed at the University of Texas Southwestern Whole Brain Microscopy Facility for embedding blocks, sections and immunofluorescence staining. For paraffin-embedded tissue, the brain samples were fixed in 4% paraformaldehyde solution in PBS then sent to the Tissue Management Core at the University of Texas Southwestern for paraffin embedding and sectioning. The monoclonal anti-calbindin-D-28K antibody produced in mouse (Sigma-Aldrich), anti-STING (Proteintech), anti-GFAP (Abcam) and anti-CD68 antibodies (Abcam) were used as primary antibodies. Donkey anti-mouse IgG(H+L) Alexa Fluor-568/647 (Thermo Fisher Scientific), donkey anti-rabbit IgG(H+L) Alexa Fluor-488 (Thermo Fisher Scientific) and donkey anti-goat IgG(H+L) Alexa Fluor-568 (Thermo Fisher Scientific) were used as secondary antibodies. Whole-brain images were captured using a Zeiss Axioscan Z1 slide scanner.

For light and electron microscopy (EM), PHAL was detected with the immunoperoxidase method, using diaminobenzidine (DAB) as a chromogen. CB immunohistochemistry for light microscopic observations relied on the immunoperoxidase method, using nickel-intensified DAB as a chromogen, while the pre-embedding immunogold-silver intensification procedure was used to detect CB labeling at the EM level.
We used a rabbit anti-PHAL (1:1000, Vector Labs, Burlingame, CA) antibody. It should be noted that no differentiated labeling was seen when the PHAL antibody was used on brain sections obtained from animals that did not receive PHAL injections. For CB, we used a mouse monoclonal anti-calbindin-D-28K antibody (Sigma-Aldrich, St. Louis, MO; catalog # C9848) derived from the CB-955 hybridoma that was produced by fusing mouse myeloma cells with splenocytes of BALB/c mice immunized with purified bovine kidney calbindin-D-28K. Western blotting revealed positive signals in cultured hippocampal neurons from rat (Chard et al., 1995 (link)) and enterochromaffin cells of the human appendix (Katsetos et al., 1994 (link)), both detected as a band with a molecular weight of 28kDa. It was tested for specificity with western blotting by Sigma-Aldrich and found not to react with other calcium-binding proteins in the EF-hand family, such as calretinin and parvalbumin.