You wrote:
We use a canine mast cell tumor as positive control - veterinary lab naturally. Probably looking for mast cells in the core. Tresa -----Original Message----- From: Bernice Frederick [mailto:b-frederick at northwestern.edu <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> ] Sent: Tuesday, June 02, 2015 12:02 PM To: Histonet at lists.utsouthwestern.edu <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> Subject: [Histonet] Toluidine blue Hello all, I was taught to do Toluidine Blue O without a control. Is there actual one and what would it be? I'm staining a bone core. Don's ask why, it's research and what a researcher wants... Plus they have a protocol they are following for this cartilaganous defect. Thanks, Bernice Bernice Frederick HTL (ASCP) Senior Research Tech Pathology Core Facility Robert. H. Lurie Cancer Center Northwestern University 710 N Fairbanks Court Olson 8-421 Chicago,IL 60611 ************************************************************************* Bernice and Tresa, Having done a bone research study like this in the past, controls should be and were carefully done. You need to know normal cartilage from treated or defect in cartilage. The researcher certainly should have set their experiment up accordingly but may have controls in place now??? You did not say if this is articular cartilage from exterior joint surfaces where they took the core or deeper in the bone at the growth plate? These two cartilages will stain differently with T blue. Normally and when studying articular cartilage defects, it is wise to also do a safranin O/fast green stain along with the T Blue. Controls are extremely important and need to be carefully set up. Hopefully, you have a contralateral bone normal core from the same animal OR a core sample from an untreated, naive control animal. It was never stated what the experimental animal model is being used? I have done a study like this in the past. When core is decalcified with an acid or EDTA, then the control needs to be decalcified exactly the same way and at the same time as experimental cores with defect. If you are decalcifying with EDTA, then you should have a normal core that is not decalcified. This is difficult with mouse but possible with larger animals. The reason is to see if the proteoglycans in the articular or even the growth plate cartilage will be extracted by EDTA, and not appreciably by buffered formic acid. Articular cartilage where proteoglycans have been removed by a decalcifying agent will have different tinctorial quality (lighter) than cartilage never exposed to a decalcifying agent. EDTA is used by biochemists to extract proteoglycans for biochemical studies, and will the same thing in a cartilage section. Hence, there will be less staining seen with the toluidine blue or the Safranin O/fast green stain after EDTA. Hence you should run two controls, 1)a decalcified cartilage control and 2) an undecalcified control. How you decalcify will be important in order to retain proteoglycans in the cartilage. I strongly suggest using buffered formic acid, available commercially. You will find recipes for buffered formic acid in text books that contain sodium formate or sodium citrate. Look for these ingredients in product MSDS before you buy the formic acid decalcifying solutions. If there is any question about EDTA versus buffered formic acid and other acid decalcifiers i.e HCl, Nitric acid, etc. for cartilage studies, I will be happy to send publications concerning this topic privately. The Toluidine blue that we do for cartilage is designed to show cartilage staining and not mast cells. It could be the mast cells might be seen along with the cartilage staining but that is not the point. The toluidine blue stain I do for mast cells is entirely different from the toluidine blue cartilage staining protocol. I will be happy to send you a toluidine blue stain procedure for cartilage and also the SafO/Fast green protocol. I have a superb T blue mast cell stain from Churukian which allows mast cells to stand out without any blue background in surrounding tissues that is often seen with other T blue staining protocols. Hope this helps. Gayle M. Callis HTL/HT/MT(ASCP) _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet