Dako envision hrp detection

Histological and immunohistochemical (IHC) analyses were performed on cats that ranged in age from 1–8 months with age-matched controls. Luxol fast blue (LFB) staining was performed as previously described [47 ] both with and without Cresyl-Echt violet counterstain. Immunohistochemical stains were performed using Dako automated immunostainer (Autostainer Link48, Dako-Agilent, Santa Clara, CA) using a low pH (6.1) antigen retrieval. IBA1 (Biocare Medical, CP290A; 1:100 dilution) and Olig2 (Abcam, EPR2678; 1:200 dilution) antibodies were incubated for 30 minutes. GFAP antibody (IR 52461–2 Dako-Agilent, Santa Clara, CA, no dilution) was incubated for 20 minutes. MAP2 antibody (Sigma-Aldrich, HM-2; 1:1000 dilution) was incubated for four hours. Detection was performed using the Dako EnVision HRP detection with DAB chromogen and hematoxylin counterstain. For quantification, slides were digitally scanned at 40x using an Aperio Scan Scope (Leica Biosystems, Buffalo Grove, IL, USA). Algorithms were written to quantify stained area of DAB for each individual IHC stain or LFB stain using Visiopharm quantitative digital histopathology software (Visiopharm, Hoersholm Denmark) and applied to all slides of an individual stain, except MAP2 as MAP2 had broad cytoplasmic staining that did not allow for individual cell density assessment.