Hi Cristina

do you looking for histological, histochemical or immunohistochemical method?

Regarding the frozen sections, are they floating or fixed?

I am just looking for quick histological recogniztion of BAT. My sections are fresh frozen cryostat sections on slides. But we can easily fixed them using Paraformaldehyde.

As you have prepared sudan black, there are two quick methods

For unfixed cryostate sections you can follow the method described in theroy and practice of histological techniques by John Bancroft:

Method (1)

1. rinse sections in 70% ethanol

2. stain for up to 2 hours in saturated Sudan black B in 70% ethanol

3. rinse in 70% ethanol to remove surface dyes, and wash in tap water

4. Counter stain (optional)

5. Wash after counterstaining and mount in glycerine jelly.

Also Bromine Sudan black method inhance the staining of sections

1. immerse sections in 2.5% aqueous bromine for 30 minutes (fume hood)

2. wash in water

3. treat with 0.5% sodium metabisulphit for i minute to remove excess bromine

4. wash in DW and stain as mentioned above

Method (2)

Sudan black 0.7 gm in 100 ml of aqueous Propylene glycol (85 ml PG + 15 DW)

1. Fix slides mounted by the frozen sections NBF or calcium formal

2. Wash in DW and drain off excess W

3. Propylene glycol, two changes, 5 minutes each

4. Sudan black, 7 minutes, agitate

5. 85% propylene glycol, 3 minutes

6. Rinse in DW

7. Counter stain in nuclear fast red, 3 minutes

8. Wash in tap water and rinse in DW

9. Mount with aqeous mounting medium.

The above method can can used also for oil red but counterstaining by hematoxylin

Good luck

H&E can also differentiate between WAT and BAT (good histologist)

Dear Cristina

Gaber Ramadan is right with H & E you can recognize the defferenza between WAT and BAT. in fact the brown fat is composed of cellular elements which present a cytoplasm full of mitochondria, the eosin colors them, while the cells constituting the white adipose tissue having a cytoplasm composed only fat and therefore not colored.

To enhance even further the differences between WAT and BAT, could be effective to perform a histological staining to highlight the mitochondrial fraction of brown fat.

1) Fixation in liquid Regaud, included in paraffin.

2) heating at 62 ° C and coloring with:

Fast dreen (Solid Green) FCF 4gr

Aniline 10 ml

Distilled water 90ml

keep this solution for 6 minutes and let cool to room temperature.

3) Place the slides in saturated aqueous picric acid with 10 minutes

4) Wash in distilled water

5) Go into a 1% solution of phosphomolybdic acid

6) Wash in distilled water

7) Coloring 5 - 6 minutes in a 1% solution of Safranin O in 50% alcohol

8) Dehydrate the slides and cover Eukitt

Best Regards

Fabio