1 Begin by placing2 the glass scintillation vial containing 3 the mouse's scBAT in PBS onto the workbench. 4 Replace the PBS with 10 milliliters of cold, 5 4%paraformaldehyde. 6 Place the vial on a nutator, 7 rocking at four degrees Celsius overnight 8 to fix the scBAT.
9 The next day, replace the paraformaldehyde solution 10 with 10 to 15 milliliters of PBS. 11 Place the vial on a nutator 12 and rock for 30 minutes at room temperature. 13 Replace the PBS with 0.85%saline 14 and continue rocking for another 30 minutes.
15 Then replace the saline with 10 milliliters 16 of 70%ethanol, 0.85%saline solution. 17 Store the vial in a four degree refrigerator overnight 18 or until ready for paraffin embedding. 19 The next day, remove the 70%ethanol saline mixture 20 from the vial and add 10 to 15 milliliters 21 of 95%dehydrant alcohol.
22 Rock the vial on a nutator at room temperature 23 for one hour. 24 After the second wash, add 10 to 15 milliliters 25 of 100%dehydrant alcohol 26 and continue rocking on a nutator for one hour. 27 To clear scBAT for embedding, add 10 milliliters of toluene 28 to the vial and continue rocking on a nutator 29 until scBAT is transparent.
30 Then embed the scBAT in paraffin wax. 31 Section the scBAT with a microtome 32 and proceed for hematoxylin and eosin staining. 33 Hematoxylin and eosin staining confirmed 34 that scBAT comprises tissue structures characteristic 35 of brown adipose tissue, 36 including numerous small multilocular adipocytes 37 in both postnatal and adult mice.
