The second immunohistochemical staining followed a different protocol, as it was performed in the Institute of Pathology of the University Hospital, LMU Munich. Instead of PBS, TRIS buffer (pH = 7.5) was used for rinsing. The dewaxing was followed by heat pretreatment with Target Retrieval Solution (Agilent Technologies, Santa Clara, CA, USA). After antigen retrieval, the endogenous peroxidase was blocked with 7.5% aqueous hydrogen peroxide. The 20 min incubation with blocking serum (ImmPRESS Reagent Kit Anti-Rabbit IgG; Vector, Burlingame, USA) was followed by a 60 min incubation at RT with the primary antibody (CCL22/MDC, No 500-P107, 1:200; PeproTech, Rock Hill, USA). The sections were then incubated for 30 min with anti-rabbit Ig (ImmPRESS Reagent Kit Anti-Rabbit IgG; Vector Laboratories, Burlingame, CA, USA). Visualization with DAB+ for three minutes and counterstaining with Hematoxylin Gill’s Formula (Vector Laboratories, Burlingame, CA, USA) followed. Aquatex (Merck, Darmstadt, Germany) was used for covering.
Immpress reagent kit anti rabbit igg
Manufactured by Vector Laboratories
Sourced in United States
The ImmPRESS Reagent Kit Anti-Rabbit IgG is a ready-to-use, peroxidase-based detection reagent designed for immunohistochemical staining. It is used to detect rabbit immunoglobulin G (IgG) primary antibodies in tissue sections or cell preparations.
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3 protocols using immpress reagent kit anti rabbit igg
1
Immunohistochemical Staining Protocol
2
Immunohistochemical Analysis of NT5C1A in PDAC
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Tissue microarrays (TMA) were obtained from the Department of Pathology, University Medical Center Goettingen (TMA-1, n = 77 patients) and the Department of Pathology, University Medical Center Erlangen (TMA-2, n = 337 patients) from postoperative PDAC patients. Written consent was obtained from every patient prior to tissue collection and analysis. Expression levels of NT5C1A were semi-quantitatively analyzed by an experienced pathologist at each site from score 0 (no expression) to score 3 (strong expression). Immunohistochemistry was performed using standard protocols with hematoxylin counterstaining. TMA-1 staining was performed with citrate buffer pH 6.0, NT5C1A antibody (Assay Biotech Cat# C15296, RRID:AB_10687827 , 1:100), and the VECTASTAIN® ABC Rabbit Kit with ImmPACT DAB Peroxidase Substrate Kit (Vector Laboratories). Pro Taqs II Antigen Enhancer pH 9.5, NT5C1A antibody (Atlas Antibodies Cat# HPA050283, RRID:AB_2681072 , 1:100) and the ImmPress Reagent Kit Anti-Rabbit IgG (Vector Laboratories) with AEC-Plus Substrate-Chromogen (Dako Denmark A/S) were used for TMA-2.
3
Immunohistochemical Detection of MSH2 and MSH6 in GBM
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Five-micrometer sections of paraffin-embedded GBM specimens were dewaxed using standard histologic procedures. Heat-induced antigen retrieval for the detection of MSH2 (1:1000; rabbit, Abcam #ab72070) and MSH6 (1:250; mouse, Dako, #3646) was carried out by boiling slides in sodium citrate buffer, pH 6.8, for 1 hour. Primary antibodies were incubated overnight at 4 °C. Specimens were then incubated with secondary antibody (Immpress reagent kit anti-rabbit IgG, Vector Laboratories, #MP7401 or Immpress reagent kit anti-mouse IgG, Vector Laboratories, MP7402) for 1 h. Bound secondary antibody was detected by ImmPact DAB substrate (Vector Laboratories, #sk-405). EGFRvIII staining was performed on a Ventana System using standard protocols (1:250, mouse, Absolute antibody, #Ab00184-1.4). Nuclei were counterstained with hematoxylin. After staining the specimes werde dehydrated covered using standard histologic procedures.
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