Question:	I wanted to know if somebody can tell me how to properly dispose of used Rapid Bone Decalcifier
Keywords	Proper Disposal, Rapid Bone Decalcifier
Answer	Used RBD (Rapid Bone Decalcifyer) can be disposed directly into sewer systems followed by a thorough tap water flush.
Question:	I have been out of the routine histology business for some time now, doing Mohs surgery as a full time job plus some. Recently my boss and I decided to start doing our own biopsies. I am pleased with the performance of your Excell Plus. I do have two questions. How do I know whether or not I can pour it down the drain? Currently I am putting it with my hazardous chemical disposal. How long can the specimens sit in the solution without making them too hard to cut. Could you let me know? Thanks, and thanks again for allowing me a normal smelling laboratory.
Keywords	Proper Disposal, ExCell Plus, Fixation Period, Archival Period, Tissue Consistency, Hard, Brittle, Tissue
Answer	Thank you for your positive comments on the performance and more pleasant (for a fixative) scent of ExCell Plus! ExCell Plus may be diluted with plenty of tap water for sanitary sewer disposal in most areas of the country including California. Because local wastewater regulations can be more restrictive than Federal and State O.S.H.A. regulations, we recommend that you contact your local wastewater authority and provide them with a copy of the M.S.D.S. for ExCell Plus and see if you can get their "blessing" for in-house disposal. We are unable to tell you exactly how long you can leave tissue in ExCell Plus before the tissue becomes too hard, but we can say that ExCell Plus does not over-harden tissue as rapidly and to the same degree as 10% Formalin. As with any tissue fixative, there is an optimum fixation time that is dependant on the type of tissue being fixed, tissue size, and tissue density. Ideally, we want to fix tissues only as long as necessary to completely penetrate and subsequently thoroughly fix the tissue.
Question:	Which of your decalcification solutions would you recommend for dissolving small (100-200 ug) quantities of 25-60 um calcium hydroxylapatite (bone) particles embedded 0.5 to 1.0 cm cubes of tissue, prior to embedding and sectioning? Specimens will be fixed in PFA or formalin and stained with H&E, Alcian Blue and/or Picro-sirius red. Tissue will be dermis or involuntary muscle (bladder/etc.).
Keywords	Bone Decalcifification, Easy Cut Deval Solution, Rapid Bone Decalcifier, R.B.D., calcium hydroxylapatite particles
Answer	We recommend using Master*Tech Easy*Cut Decal Solution, a medium strength decalcifying solution that contains both formic and hydrochloric acids. If time is a critical factor, you can also use our R.B.D. (Rapid Bone Decalcifier), a hydrochloric acid decalcifier that is compatible with the stains you will be performing.
Question:	I am just learning how to embed, section, and stain, and I'm having some trouble staining mouse knee joints with safranin O and Fast Green (to look at cartilage loss in a model of osteoarthritis). Our slides are mounted with 6um-thick paraffin sections. We use 2 changes of xylene to deparaffinze, etOH and then a rinse in dH2O to rehydrate, stain with fast green, rinse with acetic acid, stain with safranin O, then dehydrate with etOH and then 2 final changes of xylene. My problem is, the tissue is getting overstained with safranin O. Do you have any clues as to why this might be? I'm getting desperate!!
Keywords	Safranin I, overstaining
Answer	After Safranin O staining, begin the dehydration procedure with one change of 95% reagent alcohol then follow with three changes of absolute alcohol. You didn't state the strength of the Safranin O stain or how long you are staining with it; typical strength is 0.1% and staining time is usually 5 minutes. You can reduce the time in the Safranin O, as long as you get sufficient color on the cartilage. Also, are you using a nuclear stain; standard procedures include staining with Weigert's hematoxylin @ 7 minutes.
Question:	I am in the process of researching the appropriate use and PPE for the Excell Plus fixative and I'm a little confused about your statements in describing the product and the MSDS. According to the MSDS, a SCBA is required when utilizing and a hood is required when utilizing formaldehyde; I'm wondering what the difference would be. Why is this a safer product?
Keywords	ExCell Plus, Safety, Respirator, SCBA, MSDS
Answer	The reference to a self-contained breathing apparatus (S.C.B.A.) in Section 4, Fire and Explosion Information, in the ExCell Plus Material Safety Data Sheet (M.S.D.S.) specifically refers to "Special Firefighting Procedures" and not to routine handling and use. Under fire conditions, the ethanol that is a component of ExCell Plus can rapidly evaporate and become a potential hazard for firefighting personnel. ExCell Plus is considered a "safer" fixative than 10% Formalin due to the fact that it is a non-formaldehyde fixative, with formaldehyde being classified as a known human carcinogen. ExCell Plus should always be handled with appropriate care by properly trained individuals who are wearing appropriate personal protection equipment and following appropriate laboratory safety procedures.
Question:	We are using your Masson's Trichrome Staining Kit to stain myocardium frozen tissues. We followed the procedure you gave us and adjusted the time interval for better results. We are still new to this field, but the results were good so far. Our question is how often should we change each solution? So far, we don't have too many slides to stain, but in the future, we might have to do it regularly. So, how can we determine which solution needed to be replaced?? based on what criteria? Thank you very much!!
Keywords	Masson's Trichrome Stain Kit, Solution Change Frequency
Answer	The most accurate way to determine when reagents should be changed is by running an appropriate control tissue with the tissue you are staining; when the result on the control changes noticeably, it is time to change the reagents. When using a stain kit like the Masson's, some of the reagents will stain a greater number of slides than other reagents in the kit. The three reagents you need to watch most closely are the Biebrich Scarlet - Acid Fuchsin, Phosphotungstic - Phosphomolybdic Acid, and the 1% Acetic Acid. When the Biebrich Scarlet - Acid Fuchsin becomes week, muscle staining becomes less intense; however, the color of the stain solution does not change, so you have to judge its useful life by the stain intensity on the control slide. The Phosphotungstic - Phosphomolybdic Acid will get darker with use; this solution is usually changed when it becomes noticeably darker, but before it becomes opaque. The 1% Acetic Acid should be changed when it becomes medium to dark blue, although it can be used as long as the sections correctly differentiate. As the solution takes on more color, it can be harder to judge if the tissue is adequately differentiated; if you are in doubt, rinse the slide quickly in water and microscopically evaluate the result (be careful to avoid drying of the section!). If more differentiation is needed, return the slide to the 1% Acetic Acid. Thank You for using our Masson - Trichrome Stain Kit.
Question:	What is the difference between your three ion-exchange decal units? What salts are you using to remove the calcium from the bone? Will the tissue be suitable for histology after decaling in this manner?
Keywords	ION Exchange Decal Unit, I.E.D. Unit, IED Unit, Comparison, Differences, Histology, Staining
Answer	The difference between the units is just the size or quantity. Item# DCIEDEA is one 140 ml unit; Item# DCIEDCS is a case of six 140 ml units; Item# DCIEDLT is one 700 ml unit containing 5 times the volume of one 140 ml unit. Our I.E.D. Unit is comprised of a strong cation ion-exchange resin distributed in a solution containing water, formic acid, and hydrochloric acid. The combination of the acid solution and the ion-exchange resin removes the calcium ions from bone and replaces them with hydrogen ions. The decalcified tissue is rinsed thoroughly in tap water and processed in the usual manner; with the exception of an Iron Stain, all typical routine and special histological stains can be performed on the decalcified and routinely processed tissue. The I.E.D. Unit is a self-contained rapid decalcifying system that allows delicate cellular structures to remain intact while insuring that cellular detail is not compromised.
Question:	I have problem staining cell nucleus using Nuclear Fast Red. The Technical Service from Vector Laboratories thinks this is due to the interaction of Nuclear Fast Red with the treatment of the cells (iron oxide, K Ferrocyandie and CytoLyt fixative) and they suggested me to contact you for advice. I'm working with BAEC (bovin aortic endothelial cell) cultured cells. The cells containing iron oxide particles are trypsinized and then fixed in CytoLyt solution for 30 min. I then prepared cell smear on slide. So, the cell thickness on the slide is only one cell mono-layer. Here's my staining protocol: 1) 2 washes in tap water, 2 min each 2) 1% Potassium Ferrocyanide + 1% HCl for 15 min 3) 2 washes in tap water, 2 min each 4) 0.1% Nuclear Fast Red, 1 min 5) 2 washes in tap water, 5 min each 6) 70% Ethanol for 10 sec 7) 95% Ethanol for 10 sec 8) 100% Ethanol for 10 sec 9) Clear in CitriSolv, 5 min 10) Apply cover slips using DPX mounting medium I already reduced the incubation time with the counterstain from 5 min to 1 min, and increase the wash from 4 min to 10 min, but my cells are still stained pink throughout the entire cells, and the nucleus cannot be distinguished from the cytoplasm. I have also tried Neutral Red as a counterstain, but I also got similar result. I need to tell the nucleus apart from the cytoplasm for imaging purposes. Could you please advice me on this? Also, is there other counterstain better than Nuclear Fast Red for this application? Thank you very much for you help!
Keywords	Nuclear Fast Red, Nucleus Staining
Answer	While NFR is primarily a nuclear stain, it also stains non-nuclear tissue elements including cytoplasm, although cytoplasmic staining is typically very light. It may be that your cultured cells are picking up more NFR on their own or due to the other reagents you are using. You may consider diluting the NFR 1:1 using distilled or D.I. water and further reducing your NFR stain time, although 1 minute is not overly long. Although you are performing an iron stain having a blue end product, you may consider using a hematoxylin stain, which should stain only the nuclei, especially if you use a Mayer's hematoxylin at 60 to 90 seconds or a hematoxylin designed for use in IHC staining. You might also consider whether a methyl green stain, commonly used as an IHC counterstain, would serve your purpose. Methyl green will stain the nuclei without staining the cytoplasm. There are other nuclear stains, such as methylene blue and crystal violet that can be used however their color is typically more intense than hematoxylin.