Supplementary Figure 1: Antibodies validated for routinely processed tissue unpredictably stain frozen tissue sections

Effect of Antigen retrieval on immunostainability and preservation of fixed frozen sections.

A: Percentage increase in specific fluorescence over the control for antibodies against Ki-67, vimentin (VIM) and keratin 19 (KRT19) after 1, 5 and 20 minutes of Antigen Retrieval.

B: Changes in tissue preservation (tonsil) in control sections or antigen retrieval-treated sections for 1, 5 or 20 minutes. Note loss of nuclear details and partial detachment, most evident in the 20 minutes treated section. Scale bar = 100 µm

Frozen sections were immediately fixed in 4% buffered formaldehyde (FA) at RT for 18h (overnight), then washed in Tris-containing buffer for formaldehyde quenching [1]. Antigen retrieval (AR) was performed by placing the sections in a 800 ml glass container filled with the retrieval solutions (10 mM EDTA in Tris-buffer pH 8, Merck), irradiate in a household microwave oven at full speed for 8 min, followed by intermittent electromagnetic radiation to maintain constant boiling for 1min., 5 min. or 20 min., and cooling the sections to about 50Co before use. Subsequently the sections were immunostained in indirect immunofluorescence [2] with antibodies against Ki-67, Vimentin and KRT 19 counterstained with DAPI, mounted and images obtained with the S60 fluorescent scanner. To obtain a quantitative estimate of image intensity (Supplemental Fig 1A), autofluorescence background was subtracted and the submaximal fluorescence intensity calculated [3]. Fluorescence intensity for each antibody at 1, 5 and 20 minutes was expressed as percentage increase over the control. To evaluate the effect of AR on section preservation (Supplemental Fig. 1B), sections treated as above were scanned for the DAPI channel and the images were inverted with ImageJ without any further modification.

A: Widefield fluorescence imaging of fixed cells at the indicated time-points (10 min, 4h, 18h). Pixel size: 330 nm.

B: Confocal Maximum Projection of two cells at 10 min and 18 h. A cell representing the most extreme time dependent changes in the detected-antigen distribution have been selected. Pixel size: 70 nm

C: Quantification of the effect on signal intensity and distribution: signal intensity progressively decreased over time (lower graph). A completely altered and artifactual localization was detected with accumulation of the antibody in the cytoplasm and lowering of the nuclear/cytoplasmic ratio (upper graph).