Hi Cristina

Sudan Black is a stain that colors insoluble fat and lipofuscin black. So, If you are going to demonstrate residual lipid in paraffin sections, when frozen sections are not aviable, you can counter stain in nuclear fast red that staining nuclei red and residual lipids appear black. If you want to counter stain with eosin, it will stain the cytoplasm only. You should prepare sudan black in isopropyl alcohol (0.3 gm/100 cc) and 1% Borax (sodium Borate). Mix 25 cc Sudan III and 25 cc Borax. Mix, cover and filter. The stain is good for 1 day.

Good luck

Hello Gaber,

I am trying to identify patches of brown adipose tissue in non fixed frozen cryostat sections. I have prepared Sudan Black saturated in propylene glycol. Counter staining will help to have an easier interpretation. Unfortunately, we don´t have any fast red around; so, just for a test I wanted to try with things available first.

What is the role of Borax?

Thanks,

Cristina

Hellow Cristina,

Isopropyl alchol, triethyl phosphat ad propylene glycol can be used as organic solvents for Sudan stain. As mentioned in puplished articles, propylene glycol is the best one. Regarding the the role of Borax, it wil acts as a mordant. Testing eosin as a stain for the cytoplasm may or may not giving you good results. As brown fat cells have many mitochodria, eosin will stain the cytoplasm strong red as in the case of the parietal cells of stomach, but alcoholic Eosin will disolve lipids. Also, fixation with NBF or Calcium formal gives good results rather than non fixation. Finally, Osmium tetroxide protocol and ultrstructural, immunohistochemistry studies are the best for brown fat. demonstration.

Hi everybody

This is an interesting topic to follow, since I am going to stain frozen sections for detection of lipid deposits. Could it be a good solution to keep sections frozen and stain them just after preparation of the dye without fixing them? I have to point out that I will only embed samples in OCT just after sacrifice, not fix them... any recommendations, please?

thank you everybody in advance!

Manuela

I suggest the follow web: http://stainsfile.info/StainsFile/jindex.html.

Also, the method Chiffelle and Putt's Oil Red O for some Lipids in paraffin.

http://stainsfile.info/StainsFile/stain/lipid/chiffelleputtpropgly.htm

Finally Eosin Y or Eosin B dye, would be homologous and/or analogous in function to Sudan Black dye, i.e., staining the same structures, but not counter stain.

best regards,

Also, try with a trichrome stain.

To me, the Crossmon´s trichrome method is so good, to reveal cytoplasmic granules, reserves, vacuoles and cellular cytoplasm and additionally some lipids, as lipoproteins (i.e. Groat’s hematoxylin, erythrosin B-Orange G and trypan blue; Crossmon 1937, Gray 1954)

I used this method in the article: Cytological ontogeny of the digestive gland in post-hatching Octopus maya (Mollusca: Cephalopda) and cytological background of digestion in juveniles.

in this article you can see pictures in color, and the counter stain.

best regards,

Hi Mohamed

I would expose a doubt to you: has fixation with NBF to be done just after cutting sections or after cutting and drying them? Anyway, I won't stain sections before acting NBF fixation... what would you suggest?

Another question:

is liquid nitrogen-cooled isopenthane pre-treatment better than simply embedding samples in OCT, before cutting? I mean, would it lead to better results in cutting and staining sections? if not, or if the quality of the results is the same, I will continue to use the protocol suggested by Mohamed, since it's more practical for me

Thank you so much, Mohamed!

I have got some more technical questions for you:

did you have problems during cryo-cutting?

I mean, did lipid stores in samples give you problems, such as laceration of the slices?

and what about storing of the slices? could they be kept at room temperature for a long time (e.g. 1 month) or I have to store them at -20°C and drying and fixing them in NBF only when necessary?

I would be grateful to you if would you mind suggest me any published papers in which to read materials and methods, in order to have a more clear point of view on this technique...

Hello everybody,

Now, according to discussion and question of Manuela: ... suggest me any published... , that is different to original question of Cristina.

I suggest the follows journals, maybe in these is possible found some technical in this focus:

1. Anatomia, Histologia, Embryologia:

http://onlinelibrary.wiley.com/journal/10.1111/%28ISSN%291439-0264

2. Journal of Histotechnology:

http://maneypublishing.com/index.php/journals/his/

3. HISTOLOGY AND HISTOPATHOLOGY:

http://www.hh.um.es/

4. The Egyptian Journal of Histology:

http://journals.lww.com/ejhistology/pages/default.aspx

among others,

best regards,

Thank you very much to you, Omar, and to Mohamed and Cristina for this topic!

Now it's time to study and try techniques in order to reach best results for my samples!

Anyway, I'll continue to follow this topic, because I want to learn from you, too.

Best regards,

Manuela

Also, would storage of the slides be useful at -20°C for staining with dyes, if not for IHC? or do you think the best is storage at -80°C?

Mohamed, please, would you mind suggest me how to solve problems related with fat during cutting?

Hi Manuela, for long term storage (ie. a week or more) I would store them in -80. Then, just before starting the fixatoin and the treatment, take them for some minutes to -20C to cool up and from there you ca nstart. But storing them at -80 is recommmended.

Ok Cristina, thank you so much!

This is a very helpful information for me, these cryosections are too important for me to be lost with a bad storage ;-)