Process for identifying fish signals

Abstract

Among embodiments is disclosed a process for immunostaining a sample such that the stain is stable under FISH conditions.

1 . A method comprising in order:
 (a) treating a biological sample having chromosomal material therein with one or more antibodies having an affinity for at least one non-chromosomal portion of such biological sample, the antibody(ies) having an introduced non-detectable reactive conjugate thereon;   (b) treating said biological sample with a fluorescently-tagged chromosome probe having a high degree of sequence similarity to one or more portions of said chromosomic material; and   (c) treating said biological sample with a detectable tag reactive with said non-detectable reactive conjugate on said antibody(ies) but not with said chromosomal material or non-chromosomal portions of said biological sample.   
   
   
       2 . A method comprising in order:
 (a) treating a biological sample having chromosomal material therein with one or more antibodies having an affinity for at least one non-chromosomal portion of such biological sample, the antibody(ies) having an introduced a detectable reactive conjugate thereon;   (b) treating said biological sample with a fluorescently-tagged chromosome probe having a high degree of sequence similarity to one or more portions of said chromosomic material; and   (c) treating said biological sample with a non-detectable or detectable tag reactive with said detectable reactive conjugate on said antibody(ies) but not with said chromosomal material or non-chromosomal portions of said biological sample.   
   
   
       3 . A method for fixing biological material to a surface, said method comprising the steps of:
 (a) obtaining a biological sample in an aqueous supernatant and placing a least a portion of said sample on a surface to which part of the sample is to be fixed;   (b) removing aliquot volumes of said supernatant replacing the same with a similar volume of alkyl alcohol wherein said removal and replacement occurs a plurality of times so as to gradually fix the part of sample to the surface.   
   
   
       4 . The method of  claim 3  wherein said alkyl alcohol is selected from the group consisting of: the alkyl alcohol may be a C 1 -C 12  alcohol, a C 1 -C 6  alcohol, or methanol. 
   
   
       5 . A method for loading a density centrifugation gradient, said method comprising the steps of:
 (a) preparing a centrifugation gradient;   (b) applying sample to said centrifugation gradient by means of a capillary funnel.   
   
   
       6 . A method for the simultaneous identification of multiple sub-cellular components, said method comprising the steps of:
 immunostaining a sample of cells with antibodies specific to each of said sub-cellular components to be identified;   simultaneously processing said sample of cells with one or more fluorescent in situ hybridization probes comprising distinct fluorophores to discriminate between each of said sub-cellular components to be identified;   visualizing and quantitating fluorescent signals produced by said probes using a microscopy system.   
   
   
       7 . A process for identifying and enumerating fluorescent in situ hybridization (“FISH”) signals produced with respect to nuclear components hybridized in situ with fluorescent markers:
 acquiring using an epi-fluorescence microscope a plurality of images at different focal planes in each fluorescence channel corresponding to the hybridized FISH markers;   selecting a best focused image from said plurality of images for each nucleus;   acquiring using said epi-fluorescence microscope a plurality of images above and below the focal plane of said best focused image for each nucleus;   selecting for each nucleus the one focal plane above and the one focal plane below the focal plane of said best focused image in which the image is best focused;   combining said images from the one focal plane above and below said best focused image with said best focused image each nucleus to produce a combined image for said nucleus;   analyzing the combined image of each nucleus to separate background pixels from signal pixels, and to determine areas of produced signals corresponding to pre-set size and shape criteria corresponding to a non-art factual target.