Hi Wassan, you may depart the FISH procedure in a tissue-dependant first part and a probe-dependant second part. The first part comprises the pretreatment of the tissue to retrieve and permeabilises it. The amount of pretreatment is dependant on the fixation-duration of the tissue. The longer the fixation - the higher the temperature of the buffer, the longer the incubation time in the buffer, the longer the incubation time in the protease.
Troubleshooting in this part: Too low temperature of some slides because of inconsistent temperature in the waterbath. Tissueslides mounted on the glass-slides in different heights. Too short protease-incubation in relation to the fixation time. Usually nuclei can be digested until you see the first tiny holes. Underdigested nuclei are dense and look grey in the triplefilter. If the nuclei are overdigested, they get bigger holes and fuzzy borders. Different staining results due to variing fixation-times. The second part comprises the stringent wash. The temperature, buffer-quality, duration are probe-depending and are usually provided in the kit-handbook. Troubleshooting second part: Too high temp leads to loosing the bound probe. Too low temp leads to high background because of unspecifc bound probes. With your problem I would try to prolong the digestion in 10 min steps, until a good result is found. Hint: standardization of fixation is the crucial point. In our lab the tumor-block is immediately fixed in a cassette as a FISH-block. So it will be fixed very well and withstands the harsh treatment. The disadvantage is stronger autofluorescence. A further hint: be sure, that the gum around the coverslip is hardened before starting the hybridizer-protocol. 10 min drying time. I hope this helps Gudrun Lang -----Ursprüngliche Nachricht----- Von: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] Im Auftrag von wassan alkadhumi Gesendet: Montag, 23. Mai 2011 21:24 An: h n Betreff: [Histonet] FISH Dear members We just started doing FISH using HER2 FISH pharma Dx Dako kit, Code number K5331 on malignant breast tissues. I was wondering what is the success ratio or percentage for say a one run?? my hospital is the first in our region to start FISH and I am the first to run it. I have no experience with the hybridizer but by reading the hybridizer manual and the kit insert i discover it is user friendly. I run the first batch (six slides only) yesterday. Continue and finish it today. only three of the slides worked. I will start a new batch next week and I appreciate any help you may offer to avoid inaccurate result. Thank you Wassan Histotechnician Shorsh General Hospital North of Iraq _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
