Nuance fx

Protein expression and TUNEL quantitative analysis were determined using a multispectral imaging workstation (Nuance FX, Perkin Elmer, Akron, OH, USA) attached to a Zeiss Axiophot I microscope (Carl Zeiss Microscopy, Thornwood, NY, USA). Images were acquired at 40× magnification and spectrally unmixed to individual dye channels based on the spectral libraries, allowing quantitative measurement of protein expression independent of other signal intensities [36 (link),37 (link)], transformed to an expression scale from 1 (lowest) to 1000 (highest). For TUNEL assay quantitation, co-localization of the DAPI and Br-dUTP signal was determined on a per-pixel basis and expressed as a percentage.

mIHC was performed using Tyramide Signal Amplification (Akoya), imaged using the Nuance FX multispectral imaging system (PerkinElmer), and analyzed with InForm (Version 2.1; PerkinElmer) or imaged with BZ-X800 (Keyence) analyzed with ImageJ (version v2.1.0/1.53c) and Qupath (v0.2.0-m3). For confocal imaging, tissue was delipidated using the ScaleCubic protocol, immunostained, cleared with Rapiclear 1.52 (SunJin Lab), and imaged at 1,024 × 1,024 at the optimized z-resolution using Leica SP8 laser scanning confocal microscope and LASX software.

For all comparisons, serial sections were stained with the indicated method and the similar serial fields were imaged at indicated times, and under indicated conditions. All pictures were taken using a spectral camera with tunable filter (Nuance FX; Perkin Elmer) and illumination with a LED at 425nm (Cool-LED PE-2) or 435nm (Cool-LED PE-1000). LED illumination of Qdots was chosen for even, stable illumination without photobleaching of original Qdots [5 (link)].
Quantification of the intensity of fluorescence was performed using the Nuance software, with either autothreshold when different slides were compared or selection of region of interest (ROI) through automatic or manual thresholding and use of the same region to analyse the exact same areas when a time course of the fluorescence intensity was performed (slide kept on microscope stage during the whole of the experiment) or using AxioVision (Zeiss) software.