Between each step of staining the specimens were washed in distilled water or DPBS.
Assessment of percentages of positive cells was performed across the entire sample in a blinded fashion upon light microscopy by two investigators and is given as the mean of both percentages assessed in steps of 5%.Percentages of staining refer to epithelial/tumour cells within samples.
These steps ensured that the comparison of staining effect was made on the same tissues at the same position over the experimental time period.
In order to compare the intensity of staining in Mecp2-deficient mice and their respective controls, great care was taken at all stages to perform all steps of fixation, freezing, cutting, staining and scanning of the images under the same conditions, using the same solutions and timing, and by alternating tissues coming from the two genotypes.
PBS supplemented with 0.1% saponin was used in all steps of the staining procedure.
Negative controls corresponded to the omission of the primary antibodies and performing all other steps of the staining protocol.
Between all consecutive steps of the staining procedure the sections were rinsed thrice for 5 minutes in PBS.
ECM proteins laminin and collagen I were stained following tissue slice production, following the same steps of immunofluorescence cell staining.
A major advantage of this method compared to the usual techniques is the lack of purification steps and staining procedures, which often lead to cell activation.
The advantages of our approach with respect to conventional biological electron microscopy on thin sections, e.g., using immunogold labeling [5] are, 1) the possibility of imaging whole cells and 2) the absence of sample preparation steps involving staining (e.g., with compounds containing osmium, or lead), drying, and slicing [22].
