Protocol for immunofluorescence detection and quantitative analysis of pH-dependent transcriptional condensates

Authors:

Zhongyang Wu, Krishnan Raghunathan, Zhe Zhong, Jay Thiagarajah, Xu Zhou

Keywords:

IF staining; protocol; CellProfiler; FIJI; MATLAB; BRD4; transcriptional condensate;

Abstract:

Acidic pH regulates the assembly of transcriptional condensates containing BRD4 and MED1 in a variety of mouse and human cells. Here, we present a protocol to image and quantify BRD4 condensates in bone marrow-derived macrophages. We describe steps for preparing macrophage growth medium at controlled pH levels, performing immunofluorescence experiments, and acquiring images with Airyscan confocal and STED super-resolution microscopy. We detail image processing pipelines to analyze condensate properties using FIJI, CellProfiler, and a custom MATLAB program.

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