Immunofluorescence Staining in 100µm - 200µm Mouse Tumour Slices

Abstract

(from Endothelial cell death after ionizing radiation does not impair vascular structure in mouse tumor models, Kaeppler et al, 2022)

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The effect of radiation therapy on tumor vasculature has long been a subject of debate. Increased oxygenation and perfusion have been documented during radiation therapy. Conversely, apoptosis of endothelial cells in irradiated tumors has been proposed as a major contributor to tumor control. To examine these contradictions, we use multiphoton microscopy in two murine tumor models: MC38, a highly vascularized, and B16F10, a moderately vascularized model, grown in transgenic mice with tdTomato-labeled endothelium before and after a single (15 Gy) or fractionated (5 × 3 Gy) dose of radiation. Unexpectedly, even these high doses lead to little structural change of the perfused vasculature. Conversely, non-perfused vessels and blind ends are substantially impaired after radiation accompanied by apoptosis and reduced proliferation of their endothelium. RNAseq analysis of tumor endothelial cells confirms the modification of gene expression in apoptotic and cell cycle regulation pathways after irradiation. Therefore, we conclude that apoptosis of tumor endothelial cells after radiation does not impair vascular structure.

Method

Immunofluorescence staining

Mice were euthanized, and tumors were resected and immediately placed in 4% PFA in PBS and incubated overnight at 4°C. Samples were then placed into 30% sucrose in PBS solution (w/v) overnight. Tumors were embedded in optimal cutting temperature (OCT) medium, frozen in liquid nitrogen and stored at −80°C. Thin 10 μm or 60 μm cryosections were cut with a Leica CM1950 (Leica Biosystems) cryostat on glass slides (VWR). Tumor sections were air-dried, washed with PBS, and blocked with 5% BSA/5% donkey serum (v/w) in PBS containing 0.25% Triton X-100 for 1 h at room temperature in a humidified chamber. Sections were stained for proliferation with the Click-iT EdU (5-ethynyl-2′-deoxyuridine) Alexa647 immunofluorescence staining kit (Invitrogen) according to the manufacturer's instruction followed by incubation with primary antibodies in blocking solution overnight at 4°C. The following primary antibodies were used: CD31, ERG, CD45, GR1, CD68, and cleaved caspase-3 (detailed list of antibodies in Table S1). Sections were washed three times in PBS and incubated with Alexa Fluor 488-, Alexa Fluor 546-, or Alexa Fluor 647-conjugated secondary antibodies (Invitrogen, 1:500) for 1 h at room temperature in a humidified chamber. Sections were washed three times in PBS and counterstained with Hoechst 33342 (Sigma-Aldrich) and washed in PBS. Sections were mounted with ProLong Diamond Antifade Mountant (Molecular Probes). Whole tumor images were acquired with a brightfield (Nikon Ni-E) or inverted confocal microscope (Andor Dragonfly, Oxford Instruments) and processed using Imaris (Bitplane) or HALO (Indica Labs) image analysis software for spatial analysis.

For thick 100–200 μm sections, tumors were resected and fixed in 4% PFA in PBS overnight and placed in 0.25% low melting agarose in PBS solution and cut using a Vibratome (Campden Instruments). Subsequent processing of tissues was the same as for thin section.