ihc double staining  (Vector Laboratories)

Journal: Cell reports

Article Title: PARP-inhibition reprograms macrophages toward an anti-tumor phenotype

doi: 10.1016/j.celrep.2022.111462

Figure Lengend Snippet: (A and D–G) Mouse bone marrow cells were cultured as indicated in the absence or presence of olaparib or DMSO for 6 days (hereafter referred to as BMDMs), 3 donor mice for each experiment. (A) Total mRNAs were extracted and subjected to quantitative real-time PCR. Mean ± SD of three replicates, representative of three separate experiments. (B and C) K14 tumors from mice treated for 10 days with olaparib were subjected to dual-stain IHC with iNOS (red) and MAC2 (blue), scale bar = 25μm (B), and 8 fields of each slide from 6 control tumor-bearing mice and 8 olaparib treated tumor-bearing mice were counted for double positive cells. Mean ± SD, Mann Whitney test (C). (D) RNA from macrophages from 4 donor mice per experiment was differentiated ex vivo in the absence or presence of olaparib (quadruplicates for each group) and subjected to RNA-seq analysis. Pathway analysis of 500 top up-regulated genes. Green bars represent up-regulated genes in Hallmark pathways, and blue bars represent up-regulated genes in Reactome pathways. (E) Polar metabolites were extracted from BMDMs from 4 donor mice per experiment, cultured as indicated, and analyzed by mass spectrometry. Data presented as mean ± SD of 4 replicates, and comparisons were made using an unpaired t test. (F and G) Glycolytic flux is up-regulated and oxygen consumption down-regulated in olaparib-treated macrophages. BMDMs were harvested and 1 × 10 5 /well seeded in a Seahorse assay 24-well plate and cultured overnight. Extracellular acidification (F) and oxygen consumption rates (G) were determined using a Seahorse analyzer (each time point represents the mean ± SD of 5–6 cell culture replicates of bone marrow from 3 donor mice per experiment, representative of two individual experiments).

Article Snippet: Double stain IHC kit , Abcam , Cat#ab183285.

Techniques: Cell Culture, Real-time Polymerase Chain Reaction, Staining, MANN-WHITNEY, Ex Vivo, RNA Sequencing Assay, Mass Spectrometry

Journal: Cell reports

Article Title: PARP-inhibition reprograms macrophages toward an anti-tumor phenotype

doi: 10.1016/j.celrep.2022.111462

Figure Lengend Snippet:

Article Snippet: Double stain IHC kit , Abcam , Cat#ab183285.

Techniques: Recombinant, Labeling, Quantitation Assay, Staining, RNA Sequencing Assay, Mass Spectrometry, Knock-Out, Software