Rabbit anti human iba 1

Retinas, with the dorsal pole of the eyes marked with a cauterizer, were processed as previously described (Bosco et al., 2012 (link), 2008 (link), 2011 (link)). Whole-mount retinas were immunostained using mouse anti human phospho-neurofilament (pNF, Dako, Carpinteria, CA), rabbit anti-human Iba1 (Wako, Richmond, VA), goat anti-GFP (Abcam, Cambridge, MA), and rat anti-mouse CD169 (sialoadhesin) conjugated to Alexa Fluor 647 (MOMA-1 clone, AbD Serotec, Bio-Rad, Raleigh, NC) primary antibodies, which were detected with Alexa-Fluor-conjugated donkey anti-IgG secondary antibodies (Invitrogen, La Jolla, CA).

The brains were harvested and immersed in 4% paraformaldehyde (Fisher) overnight. Then, brains were washed 3X with sterile saline for 1 h, embedded in paraffin, 4 μm coronal sections were serially cut using a cryostat (Tanner Scientific, model: TN50), fixed onto glass slides, and subjected to hematoxylin & eosin or Periodic acid-Schiff staining to examine tissue or fungal morphology, respectively. GXM (MAb 18B7 is an anti-cryptococcal GXM IgG1 generated and generously provided by Arturo Casadevall at the Johns Hopkins Bloomberg School of Public Health; 1:1,000 dilution) and Iba-1 (rabbit anti-human Iba-1; 1:1,000 dilution; FujiFilm Wako) specific Ab (conjugated to horseradish peroxidase; dilution: 1:1,000; Santa Cruz Biotechnology) immunostaining to assess capsular release and distribution and microglial phenotype, respectively, near cryptococcomas. The slides were visualized using a Leica DMi8 inverted microscope, and images were captured with a Leica DFC7000 digital camera using LAS X digital imaging software. GXM distribution in tissue sections at 10X magnification (n = 15 fields per brain) was calculated using NIH Image J color deconvolution tool software (version 1.53q). The mean color intensity of the GXM for each treatment group was plotted in Prism version 9.5 (GraphPad). The images were examined and analyzed by Dr. Mohamed F. Hamed, a veterinary pathologist.