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Host species: Horse
NB: Sucrose- infiltration serves to minimise ice-crystal artefact but not eliminate it if freezing is far too slow; it is not an essential step.
Before immunostaining, the 8 micron sections were placed in a 60degreeC oven for 60 mins to enhance adhesion (cryosections from PFA-fixed tissue have a tendency to come off slides, even Superfrost Plus/subbed or PLL-coated slides (in my experience). Before incubation in primary Ab,I blocked endogenous peroxidases and also biotin. I used a biotinylated secondary Ab that had been rat-absorbed to ensure that endogenous rat Immunoglobulins would not be positive.
Submitted image shows white pulp (PALS) and a small area of red pulp (RP-upper left). The Periarteriolar Sheath (PALS) with it's Central Arteriole/artery (CA)shows many positive cells (B-lymphocytes and Macrophages) and negative lymphocytes (T-cells?). The Marginal Sinus (MS) is clearly seen between the PALS and the Marginal Zone (MZ). There is a clear Germinal Centre (GS) in this image.
Lymphoid tissue is not my speciality so I apologise for any inaccuracies.
NB: for the attention of any who are concerned that my 60degreeC pretreatment of sections adversely affected positivity: I had previously cut/immunostained sections that had been dried at RT for two hrs, under a fan. These sections gave identical immunostaining but, much of the section had become dislodged from the slide.
MR. Carl Hobbs
Submitted Apr 27 2010