Immunohistochemical Staining of Sialic Acid and PECAM-1

Tissue was deparaffinized for 20 min at 60 °C; Diva Decloaker, 20X (Biocare medical, CA, USA) and Decloaking chamber™ NXGEN were used for recovery. The sections were incubated with primary antibody (Polyclonal Antibody to Sialic Acid, abx 100,414, abbexa Ltd. Cambridge, UK), and PECAM-1 (Polyclonal Antibody Abbiotec 250,590) diluted in blocking serum and incubated for 48 h at 4 °C, then washed 3 times with TBST, for 3 min each. They were then incubated with MACH 2 Double Stain 1 polymer (Biocare Medical MRCT523, CA, USA) for 30 min and washed with TBST. Diaminobenzidine DAB (Biocare Medical, CA, USA) was used for development. A negative control, which had no primary antibody, was performed in all groups, and salivary gland tissue was used as control tissue. IHC quantification of sialic acid was performed with ImageJ software (https://www.rsbweb.nih.gov/ij) developed by the National Institute of Health (NIH), using the IHC Profiler plug-in for the quantitative analysis of immunohistochemistry samples [16 (link)].
For evaluation of CD31/PECAM-1 immunohistochemical staining, slides were observed under a light microscope, and positivity was determined in ten microvessels. [17 ]