Slides were de-paraffinized in xylene and rehydrated through graded ethanol followed by heat mediated antigen retrieval using 10 mM Sodium Citrate Buffer (pH 6.0). IHC was performed via incubations with one primary antibody followed by host-matched secondary polymer reagent and color substrate. Primary antibodies used were as follows: CD3+, CD8+, HLA-DR, CD68 (A0452, M7103, M0746, M0814 – Dako), CD163, Ki-67, and αSMA (ab189915, ab16667, ab5694 – Abcam), cleaved-Caspase-3 (#9661, Cell Signal Technologies), and FOXP3 (14–4777–82, eBioscience). Secondary reagents were ImmPress Rabbit HRP and Mouse HRP (Vector Laboratories). Color development was performed using Quanto DAB brown (Fisher Scientific). Slides were counterstained with Harris hematoxylin (Sigma) as appropriate, dehydrated and mounted.
Quanto dab brown
Manufactured by Thermo Fisher Scientific
Quanto DAB brown is a chromogenic substrate for immunohistochemical detection of antigen expression. It produces a brown precipitate at the site of the antigen-antibody reaction, allowing for visualization of target proteins in tissue samples.
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Lab products found in correlation
2 protocols using quanto dab brown
1
Immunohistochemical Profiling of Tissue Samples
2
Immunohistochemistry of Formalin-Fixed Paraffin-Embedded Tissue
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Formalin-fixed paraffin-embedded tumor slices were cut into 4 uM sections and placed on slides. Slides were deparaffinized in xylene and rehydrated through graded ethanol followed by heat mediated antigen retrieval using 10 mM Sodium Citrate Buffer (pH 6.0). IHC was performed via incubations with one primary antibody followed by host-matched secondary polymer reagent and color substrate. Primary antibodies used were as follows cleaved-Caspase-3 (CC3; #9661, Cell Signal Technologies) Secondary reagents were ImmPress Rabbit HRP and Mouse HRP (Vector Laboratories). Color development was performed using Quanto DAB brown (Fisher Scientific). Slides were counterstained with Harris hematoxylin (Sigma) as appropriate, dehydrated and mounted. Images of stained slides were obtained using Hamamatsu Nanozoomer whole slide scanner at the University of Washington Histology and Imaging Core. Quantification of IHC was not performed due to limitation in tissue for analysis.
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