Immpact dab eqv peroxidase hrp substrate

Formalin-fixed paraffin-embedded sections were deparaffinized with xylene and then rehydrated with gradient ethanol and water. Antigen retrieval was conducted with 70% formic acid followed by heating in a pressure cooker (Aptum Bio Retriever 2100, Aptum Biologics Ltd, UK). Endogenous peroxidase activity was quenched with 1% H₂O₂ in 50% ethanol for 30 min and then washed in distilled water. Sections were further blocked with 5% normal horse serum prior to incubation in medium containing primary antibody phospho-S129 (Abcam Ab51253, 1:500) or O2 (Creativebiolab TAB-0748CLV, 1:200) at 4 °C for 2 days. Sections were washed and incubated with either alkaline phosphatase (AP)-anti-mouse or horseradish peroxidase (HRP)-anti-rabbit secondary polymer antibodies (VECTOR, ImmPRESS, cat.# MP-5402 & MP-7401). The immunoreactive color was developed with ImmPACT Vector Red AP Substrate (VECTOR, SK-5105) or ImmPACT DAB EqV Peroxidase (HRP) Substrate (VECTOR, SK-4103) respectively, prior to counterstaining with hematoxylin. Sections were then dehydrated with gradient ethanol and cleared with xylene prior to coverslipping with D.P.X. Images were obtained at × 20 magnification using an Olympus slide scanner (VS120).

Flag was detected using a Mouse on Mouse (MOM) ImmPRESS HRP (Peroxidase) Polymer Kit (Vector Laboratories, MP-2400). Paraffin-embedded livers were cut into 5 μm sections, deparaffinated in xylene and rehydrated in a series of graded alcohols and water. For antigen unmasking, sections were incubated in citrate-based antigen unmasking solution (pH 6.0; Vector Laboratories, H-3300) at high temperature for 20 min. After blocking in BLOXALL Endogenous Blocking Solution (Vector Laboratories, SP-6000) for 10 min and a 1 h incubation in MOM Mouse IgG Blocking Reagent, sections were stained with mouse monoclonal anti-DYKDDDDK Tag antibody (1:100; Cell Signaling Technology, 8146) in 2.5% normal horse serum MOM solution overnight at 4 °C. DYKDDDDK signal was revealed by incubation with MOM ImmPRESS Reagent for 10 min and enhanced with ImmPACT DAB EqV Peroxidase (HRP) Substrate (Vector laboratories, SK-4103) for 1 min. Sections were counterstained with haematoxylin, dehydrated, mounted with Permount mounting medium (Fisher, SP15) and imaged in a Zeiss Axiolab 5 microscope/Axiocam 305 colour camera (Carl Zeiss Microscopy). Quantification of Flag percentage area was performed as described⁷⁸ .