The development of R7 axons, particularly with regards to their layer specificity (Ting, 2005; Lee, et al., 2001; Clandinin, et al., 2001) and columnar restriction (Ting et al., 2007; Ting, et al., 2014) has been studied extensively in fixed preparations.
The class of asymptotic nonexpansive mapping have been extensively studied in fixed point theory since the publication of the fundamental paper [1].
The results clearly show that even highly abundant proteins, like Hsp70, are only poorly detectable using EXB, if the sample was fixed extensively.
Cells were then washed extensively with PBS, fixed with 4% paraformaldehyde, stained with DAPI, and fluorescent images were acquired using the Olympus FV10i confocal microscope (Olympus Corp., Tokyo, Japan).
The colon rectum was opened longitudinally and washed extensively with PBS, fixed in 4% buffered formaldehyde for 24 h and then stained with methylene blue.
The organ was opened longitudinally and washed extensively with PBS, fixed in 4% buffered formaldehyde for 24 h and embedded in paraffin.
Following incubation with secondary antibodies (30 min, RT), FITC-conjugated anti-mouse IgG (1 : 80) and TRITC-conjugated anti-rabbit IgG (1 : 400), the cells were washed extensively in PBS, fixed in Dapi, mounted with Hydromount and visualised by microscopy on a Nikon Eclipse E600 microscope.
After overnight incubation at 37°C in a humidified CO2 atmosphere, the PBMCs were removed, and the adherent cells extensively washed, trypsinized and fixed for FACS analysis.
The masses were debrided extensively and tissue samples fixed in 10% neutral buffered formalin, embedded in paraffin wax and sectioned at 4 μm.
Proper fixed samples were extensively washed in ethanol 70% (3 × 24 h) to remove fixative, and then, the specimens were dehydrated in ascending graded concentrations of ethanol (80, 95, and 100%), cleared in xylene, and embedded in paraffin wax.
