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Where there was doubt about the intensity of the staining, reference was made to photographs that had been prepared as standards.
Whenever a staining procedure was performed, negative and positive control tissues were simultaneously stained with new battery of tissues and then the control tissues were used as a staining reference.
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The volume of interest was calculated by quantification of the volume of staining referenced to the surface area of basal lamina surveyed (cubic micrometre per square micrometre).
Briefly, after standard deparaffination of the tissue sections, the microdissected tissue of interest (as indicated in the haematoxylin/eosin stained reference sections) was transferred into EXB Plus buffer.
The nc82 channel for each specimen was registered with an nc82 stained reference brain that includes the MB calyx and LH.
The results perfectly agree with HE stained references slides prepared separately making multiplex CARS an ideal approach for chemoselective imaging.
The results were compared to brightfield images of hematoxylin and eosin (H&E) stained references and to lipid intensity contrasted images of MCARS raw data.
We examined the pattern of Nissl staining with reference to published anatomical guides [1], [76] in order to evaluate the lesions.
All areas of immunoreactivity were aligned to protein spots on equivalent silver stained 2D reference gels (Figure 1b) and these protein spots were subsequently identified using nano-LC MS/MS.
The staining was scored as 0 (less than 10% positive cells), 1 10-200% positive cells), 2 21-500% positive cells), 3 (more than 50% positive cells) based on reference staining slides using a high magnification (x400).
Therefore, we proceeded to visualize how staining patterns of styryl molecules were related to the staining patterns of specific reference compounds.
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