Comprehensive Tumor Immunohistochemistry Analysis

Tumors from both s.c. and orthotopic efficacy studies were formalin-fixed, embedded in paraffin and subjected to immunohistochemical analysis via staining with antibodies directed against CD8 (Abcam, ab203035), αSMA (Santa Cruz Biotechnology, A1218), pSTAT3 (Cell Signaling Technology, Inc., 9145L), CD11c (Abcam, ab33483), CD45R/B220 (Fisher Scientific, BDB557390), and F4/80 (Abcam, ab100790). For analysis, images were acquired at 10x and 20x magnification (approximately 5-25 pictures per mouse depending on the size of the tumor) and captured using NDP.View2 software (Hamamatsu Photonics K.K., Japan). Pictures for the staining of pSTAT3 and CD8 were analyzed by Fiji ImageJ (GitHub) to quantify the percentage of positive cells or the count of the positive cells. CellProfiler (Broad institute, Cambridge, MA) was used to analyze the αSMA staining pictures. (28 (link),29 (link)) Qupath, was used to quantify the staining for CD11c, B220 and F4/80. Orthotopic tumors were also stained for DAPI (Perkin Elmer), CD4 (Abcam, ab183685) and FOXP3 (Novus Biologicals, NB100-39002) and then visualized using a Vectra Polaris immunofluorescent whole slide scanner. Qupath was used to quantify the number of CD4⁺FOXP3⁻ and CD4⁺FOXP3⁺ cells per cm² tissue. (30 (link))

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Zhang Y., Ware M.B., Zaidi M.Y., Ruggieri A.N., Olson B.M., Komar H., Farren M.R., Nagaraju G.P., Zhang C., Chen Z., Sarmiento J.M., Ahmed R., Maithel S.K., El-Rayes B.F, & Lesinski G.B. (2020). Heat shock protein-90 inhibition alters activation of pancreatic stellate cells and enhances the efficacy of PD-1 blockade in pancreatic cancer.. Molecular cancer therapeutics, 20(1), 150-160.

Publication 2020

ab100790 NDP.View2 software ab183685

Antibodies Biologicals Cd4 cells Dapi Embedded paraffin Formalin Immunofluorescent Mouse Novus Tissue Tumors Vectra αsma

Corresponding Organization :

Other organizations : Emory University, First Affiliated Hospital of Xi'an Jiaotong University, University of Illinois Chicago

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Variable analysis

independent variables

Treatment (antibodies directed against CD8, αSMA, pSTAT3, CD11c, CD45R/B220, and F4/80)

dependent variables

Percentage of positive cells or count of positive cells for pSTAT3 and CD8 staining
Staining for αSMA
Staining for CD11c, B220 and F4/80
Number of CD4+FOXP3- and CD4+FOXP3+ cells per cm2 tissue

control variables

Formalin-fixed, paraffin-embedded tumors from both s.c. and orthotopic efficacy studies
Images acquired at 10x and 20x magnification (approximately 5-25 pictures per mouse depending on the size of the tumor)
Analysis software used (Fiji ImageJ, CellProfiler, Qupath, Vectra Polaris immunofluorescent whole slide scanner)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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