Immunofluorescence of human metastatic lymph node

Description:

Immunofluorescence staining of a consecutive, 10 µm-thick section (IF section #1), consecutive to Open-ST metastatic lymph node section #2

Data is provided as a standard, non-compressed pyramidal OME.TIFF file with 3 channels, converted with QuPath.

Channels:

0 (cyan): DAPI1 (yellow): panCK2 (magenta): VIM

Methods:

Immunofluorescent (IF) staining was performed on the first cryosection of the metastatic lymph node reserved for validations, as shown in the experimental setup in Figure 2D. This slide was reserved at -80°C for ~15 months, before proceeding to IF staining. Steps were performed at room temperature unless stated otherwise. Upon drying the slide, the OCT was removed in a 10-minute PBS wash. Next, the section was fixed with 4% formaldehyde (Sigma-Aldrich, F8775) for 15 minutes and then washed with DPBS (no calcium, no magnesium, Gibco™, 14190169) three times. Blocking and permeabilization was done by incubating in 0.25% Triton-X (Sigma-Aldrich, T8787) and 5% normal donkey serum (Biozol Diagnostica, SBA-0030-01) in DPBS for 1 hour. 

The section was incubated overnight at 4°C in the dark, with primary-conjugated antibodies diluted in a DPBS buffer with 0.1% Triton-X and 5% normal donkey serum, as follows: 1:100 for Pan Cytokeratin (mouse mAb, clones AE1and AE3, eFluor™ 570 conjugate,  ThermoFisher, Cat# 41-9003-82), 1:50 for Vimentin (mouse mAb, clone V9, Alexa Fluor® 750 conjugate, Bio-Techne, Cat#  NBP1-97670AF750). Following three 5-minute DPBS washes, DAPI staining was performed with 1 ug/mL DAPI (Bio-Trend, #40011) in DPBS for 10 min in the dark. After three DPBS rinses, the section was dried and then mounted with 85% glycerol. 

Images were acquired on the Leica Thunder DMi8 imager using a Leica DFC 9000GT sCMOS fluorescence camera, a 20X objective and the Leica Application Suite (LAS) X software (v.3.9.0.28093). Thunder instant computational clearing was performed on the image.