Hello All, I have compiled all of the responses to date regarding the question I posted on histonet about too much volume and reverse capillary action: (I've changed font styles to differentiate between responses)
I have used them for quite some time. I was having problems with uneven staining and determined that it was from the being too full and was drawing up liquid from the bottom. There is a small opening on the end and I just empty the solution from it. I usually empty it after rinsing the antibody off and then usually before putting DAB or DAPI on depending on the number of slides stained. As for washing, I just wash in a solution of soapy water and then rinse in distilled. I use the coverplates for several staining times. I love the sequenza and prefer it to having an automatic stainer. I still use these and have 9 racks. The coverplates can be found on eBay, but I clean them in soapy bleach water, rinse well, and dry until the plastic gets brittle and they break. I use AEC 99% of the time so I do not generate the waste of DAB. If the racks are filing up with reagents and solutions, leave an empty slot and pipet it out. Otherwise, I dump it and and rinse after the run. For the antibodies and detection solutions, you only need 3-4 drops. I only fill the well to the top with rinses. We use these units, I have never had an issue with the waste reagents touching the bottom of the plates. We never use more than 3 drops of kit items, the wells are filled with buffer. It never seems to be alot of waste. After we finish staining we just rinse the holders out with running water for a few minutes and let air dry. We empty them before they get to that stage, as this would be bound to affect the efficient working of the Coverplates. This is Tyler Liebig with the Thermo Scientific IHC group. I'm responding to Keri's questions regarding the Sequenza manual staining. Keri you are correct that if the waste is not emptied and builds up too much in the bottom of the sequenza unit it will definitely stop the capillary action (Reagent can't flow both ways :-). However, this can be avoided by emptying the waste regularly between runs. You bring up a good question though that I didn't know the answer to: "How many slides can be stained before the waste hits the slides?" I did a quick calculation below that I hope is helpful. Total Waste Volume of Sequenza Rack: In a Sequenza rack the slides are held about 1.5 inches above the bottom of the tray. This allows about 270ml of waste before the level is close touching the slides. Reagent use per slide: If you stain a single slide with a long protocol then worst case scenario is about 7 steps. (H2O2 through Counterstain with a two step polymer) Volume of rinse buffer: This means you would rinse 8 times 2ml each (16ml total rinse buffer per slide worst case). For the staining reagents: The recommended volume is 0.1ml per reagent and 0.5ml for chromogen. So for 6 reagents plus chromogen that is 1.1ml and I will double it because I know some people add way more than needed. So 2.2ml per slide worst case. So to sum it up worst case scenario 18.2ml of waste is created per slide which means 14 slides (270/ml/18.2ml=14.8) can be stained before the waste risks touching the slides. The rack holds ten slides total. In the end I recommend users play is save and empty the waste after each batch of slides stained even though most user generate much less waste than the calculations above. I also included a link to a great description of Sequenza use on the Histonet posted by Gayle M Callis HTL/HT/MT (ASCP). I have referenced these details frequently myself (Thanks Gayle). http://www.histosearch.com/histonet/Jul02A/Longansweronusetechnicswi.html I have also read through Gayle's post on Histonet, it is worth reading through for some more insight on the use of these units. Keri _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
