Four-µm sections of paraffin slides obtained from patients with PDAC were provided from the pathology department. Primary antibodies—anti-CD3 (ab5690), anti-CD8 (ab17147), anti-CD20 (ab78237), anti-CD66b (ab197678), and anti-α-sma (ab5694)—were purchased from Abcam PLC (Cambridge, UK). A Masson-Goldner staining kit (100485) for collagen staining was bought from Merck KGaA (Darmstadt, Germany). Secondary antibodies—horse anti-mouse IgG (BA-2000) and horse anti-rabbit IgG (BA-1100)—were purchased from VECTOR Laboratories (Burlingame, CA, USA), along with the Avadin/Biotin Blocking Kit (SP-2001), the alkaline phosphatase enzyme detection system (ABC-AP) (AK-5000), and the ImmPACT Vector Red Alkaline Phosphatase Substrate Kit (SK-5105).
Abc ap ak 5000
Manufactured by Vector Laboratories
Sourced in United States
The ABC-AP (AK-5000) is a laboratory instrument designed for general-purpose applications. It is capable of performing a range of analytical tasks, but a detailed description of its core function cannot be provided while maintaining an unbiased and factual approach.
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2 protocols using abc ap ak 5000
1
Immunohistochemical Analysis of PDAC Samples
2
Immunohistochemical Staining of CD45 Leukocyte Marker
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Pan-leukocyte marker CD45
The anti CD45 (BD Pharmingen, # 550539) was diluted 1:25, isotype control antibodies (rat IgG2b 1:400; Biolegend, # 400622) served as a negative control. Depara nised sections were heated in a microwave oven (800W) in DAKO retrieval solution (Target Retrieval Solution DAKO by BIOZOL, Germany # S1699). Slides were incubated 1 h at room temperature with the primary antibody, rinsed and then incubated for 30 min at room temperature with the respective secondary rabbit α rat-biotinylated antibody (Jackson Immuno Research # 312-065-048). CD45 antibody binding sites were detected by incubation of the sections with the streptavidin-alkaline phosphate complex (ABC-AP # AK-5000; Vector Laboratories, Burlingame, CA, USA) for 30 min. For visualizing enzyme reactivity of the alkaline phosphate complex, liquid permanent red (DAKO by BIOZOL, Germany # K0640) was applied to the sections for 15 min under visual control. Then they were washed again under running tap water and nally rinsed in distilled water.
After counterstaining with hemalaun, they were nally mounted using a resinous permanent mounting medium (Eukitt, Kindler GmbH, Freiburg, Germany).
The anti CD45 (BD Pharmingen, # 550539) was diluted 1:25, isotype control antibodies (rat IgG2b 1:400; Biolegend, # 400622) served as a negative control. Depara nised sections were heated in a microwave oven (800W) in DAKO retrieval solution (Target Retrieval Solution DAKO by BIOZOL, Germany # S1699). Slides were incubated 1 h at room temperature with the primary antibody, rinsed and then incubated for 30 min at room temperature with the respective secondary rabbit α rat-biotinylated antibody (Jackson Immuno Research # 312-065-048). CD45 antibody binding sites were detected by incubation of the sections with the streptavidin-alkaline phosphate complex (ABC-AP # AK-5000; Vector Laboratories, Burlingame, CA, USA) for 30 min. For visualizing enzyme reactivity of the alkaline phosphate complex, liquid permanent red (DAKO by BIOZOL, Germany # K0640) was applied to the sections for 15 min under visual control. Then they were washed again under running tap water and nally rinsed in distilled water.
After counterstaining with hemalaun, they were nally mounted using a resinous permanent mounting medium (Eukitt, Kindler GmbH, Freiburg, Germany).
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