| From: | kevin williams |
Here are all the responses I had to the three questions I asked. So thanks for all the help.
Processor: – Changing Solutions
I would recommend rotating solutions with fresh ethanol and xylene in the last containers and fresh 10% formalin and 70% ethanol (if you use these) once/week. Wax is a little hardier, I would rotate and add fresh wax in the last container once/fortnight.
I have 2 Sakura VIPs and am so pleased. We rotate reagents after 150 cassettes and do a total change out after 300; and we have excellent quality. This may be an overkill, and our tissues are pretty clean (not bloody or other precipitate); so, I think you can do a little adjusting and you will be OK.
Theleria Hackett, B.S., HT (ASCP)
The suggestion of rotating the formalins, alcohols and clearant is the best way to go weekly. You should replace the 70% when it appears to be cloudy or dirty. Depending on your tissue load this could be twice a week. As far as paraffins they should be rotated or checked weekly also. If you can smell xylene or clearant strongly you have gone too long. Paraffin with high amounts of clearant will not infiltrate well and sectioning can be a nightmare. The tissue will cut like dry cheese or in some cases very smelly with clearant in the block. The sections will fly apart on the water bath or just fall apart on the knife.
We also have a Sakura VIPs. We process about 50-200 blocks per day. We change our processor once a week when we have a big week of fatty tissues (800-1000). When I worked in a research lab, we did about 10-25 blocks per day. Generally less than 100 blocks per week. With the volume of fluid the VIP containers hold, we found we only needed to rotate the solutions every two or three weeks. But we cleaned and changed the process on the first Monday of the month...just to be on the safe side. Our processing was always good. Manual Immunos: We're the Cell Marque representative for Argentina. I suggest you to contact the Latin America representative, Alfonso Heras. He's the owner of BioSB in Santa Barbara, CA. Please take a look at his Website http://www.biosb.com He's promoting a manual system for IHQ and ISH that uses capillary technology. Biocare Medical sells an IQ stainer that is very affordable and really limits the hands on for IP’s. I have lots of experience with it and have nothing negative to say about it or their company. Good Luck! Kevin, there is a hand method sold by Fisher using probe on capillary gap slides that can do 10 -20 at once. It is manual but it has a 37 degree chamber to incubate. It is a good solution to total manual, the heat speeds up the reactions. It has been many years since I have done this but if you contact Fisher about Histology products you may find thus system, it is quite small too. Best of luck and call me anytime. Here is the method we use M719601 Dako primary antibody 1:150, Envision mouse 30:30 incubation, citrate AR. Regards Have a look at the Shandon Sequenza system............... Richard Edwards.....................u.k................ Try Shandon..they have a great counter top manual staining system!! Give me your fax# and I'll fax you a copy of our protocol for "Mel A". (I'm assuming you are talking about Melan A or Mart-1). You might want to talk to your pathologist about using a melanoma cocktail (a mixture of Melan A and HMB-45) from Biocare Medical. We have found that it is far superior to either alone. If you speak to Chris Von Vedder, (800) 799 9499 and tell her you know me, I'm sure she'll send you a sample. What kind of equipment do you have for antigen retrieval? Kevin, Cell Marque caters to small volume labs. We do not manufacture a machine, so we have smaller volume antibodies to serve the volumes and budgets of smaller labs. We also make it easy for people to start up with immunos by developing an inexpensive, standardized pretreatment that allows you to heat retrieve your slides in 20 minutes. And Cell Marque does have Melan-A, as well as the less specific S-100 and HMB-45, and also the MORE specific Tyrosinase. I am attaching a sheet showing the price per slide of using Cell Marque products. If you have any questions, you can contact us at 1-800-665-7284. Estimated start-up costs for immunohistochemistry staining based on Cell Marque product pricing: Pretreatments range from $.17/slide to $.39/slide (figures based on 20 slides/200 ml) Antibodies range from $1.33/slide to $7.90/slide Detection ranges from $.65/slide (large detection kit) to $1.72/slide (smallest Kit) $2.15 to $10.01 per slide with Cell Marque products Xylene and substitutes: No sir, I've been doing it for a long time, Xylene and Citrisolv (subs) in processor. Xylene in stain line and coverslipping Could you clarify this question please? Do you mean you want to deparaffinize with a xylene substitute after sectioning? It is unclear what you mean by embedding station. If you are using xylene on the processor and want to deparaffinize, stain and then coverslip from a xylene substitute there should be no problem. You will need to extend times in both the processor and deparaffinization to get good clearing/infiltration and paraffin removal with the d-limonene based products. Pam Marcum
The paraffins?....we did the "finger check" on paraffin #3. You stick in your index finger, coat it with the paraffin, and rub it with your thumb. If the paraffin is slimy, it is time to discard the first two paraffins, rotate the l
I highly recommend polymer detections like Dako's EnVision. Fast and easy.Please take a look at http://home.no.net/immuno
Kathy Bowden HT