Paraffin embedding of adipose tissue

The isolated (brown) adipose tissue was collected in PBS and transferred to PFA solution (4%
PFA/PBS) and incubated for 30 min at room temperature (RT). Next, tissue samples were
dehydrated by subsequent washes in ethanol of ascending concentrations (50%, 70%, 80%,
90%, and 100%) for 1 h each, incubated 2 times in xylol for 30 min and placed in paraffin
solution 3 times for 1 h at 55° C. The tissue was placed in embedding forms and was
embedded with fluid paraffin. Paraffin blocks were stored until cutting at 4° C. Paraffin
blocks were cut in 4 μm thick sections using a microtome (Microm). Quality and orientation
of the tissue was frequently checked under a light microscope (Leica). Slides were dried at RT
for 1 - 2 h and finally stored at 4° C.

H&E staining
In order to perform a hematoxylin/eosin stain, paraffin sections were treated 2 times for 2
min in xylol (deparaffinization) followed by incubation in 100%, 90%, 80%, 70%, 50% and
PBS for 2 min (rehydration). Slides were then treated for 1 min with hematoxylin (Mayers
hemalaun) and blued in tap water. Subsequently, slides were stained with eosin for 1 min and
washed again in tap water. Sections were dehydrated in 50%, 70%, 80%, 90%, 100%, ethanol
for 2 min each, washed 2 times 5 min in xylol and finally mounted with Roti®-Histokitt.

Mayers hemalaun, Merck (Cat.No. 1.09249)

Eosin G, Merck (Cat.No. 1.09844)