Metaphase Spread: Telomere/Centromere-directed FISH
Slides should be dropped the day before following the protocol.
Prepare fresh solutions of:
1)
2)
3)
4)
5)
25%, 50%, 100% EtOH
4% Formaldehyde in PBS
Acidified Water (39.6ml mQH2O + 40μl concentrated HCL), ph2. Keep at 37oC.
PBS
Thaw 1ml aliquot of pepsin (100mg/ml)
1) Rehydrate slides for 15min in PBS.
2) Fix for 2min in 4%Formaldehyde in PBS.
3) Wash for 1min in PBS, then dip briefly through ascending EtOH series (25%, 50%, 100%).
Transfer slides into 25% from PBS, replace lid, shake, transfer slides into 50%, replace lid,
shake, transfer slides into 100%, replace lid, shake. Transfer slides into slide drying rack and air
dry for 10min minimally or until completely dry.
4) Add 400μl of pepsin stock to acidified H2O. Place slides into pepsin solution and leave in
37oC water bath for 15min. Shake Coplin jar vigorously every 5min. After 15min check slide
under light microscope to ensure complete cytoplasmic digestion. If not digested add more
pepsin and continue digestion, check every 5 min.
While samples are digesting prepare the probe solution. See Solutions.
5) Once digested follow step 3. PBS wash and EtOH series.
6) Add 100μl of working probe solution to each slide, cover with coverslip and denature in
hybridization oven for 15min at 95oC.
7) Transfer slides into 37oC humidified incubator and leave o/n. Approximately 16hrs.
8) Remove coverslips and wash slides with Formamide Buffer for 15 min (X4).
9) Wash slides with Tween 20 Buffer for 5 min (X4).
10) EtOH series and air dry.
11) Drop of Vectashield + Dapi on each slide, cover with coverslip, and store at –20oC.
Solutions
Blocking reagent.
Dissolve 5g blocking reagent (Sigma cat# 1096176) in 50ml (150mM Maleic acid, 150mMNaCl,
ph7.5 (adjusted with NaOH)) on heating block. Autoclave and store at 4oC.
Formamide Buffer
8ml 1M Tris-HCl ph7.8
232ml mQH2O
520ml Formamide
This needs to be prepared at least a day in advance. The buffer needs to be at RT for washing
but the formamide is stored at 4oC, therefore making as needed will not be at the correct
temperature.
70% Formamide Buffer with Blocking Reagent
10ml blocking reagent
90ml Formamide Buffer
Tween 20 Buffer
1L PBS ph7.2
250μl Tween 20
Probe
Probes are stored at 500μg/ml in TE. Per slide working solution for telomere probes (67Cy3,
67F, 67FG) is 0.4μl probe per slide, centromeric probe (104F, 104R) is 0.8μl per slide, 1μl 1M
MgSO4 , up to a total volume of 100μl with 70% Formamide Buffer with Blocking Reagent.
Quantification
1) Images captured using Hamamatsu Orca camera, 12 bit color depth, mounted on a Zeiss
Axioplan 2 fluorescence microscope.
2) Images captured using Openlab 2.2.5 software as detailed below.
2.1) Image on field selected using dapi filter and focused to best point.
2.2) Saturation set at one level and exposure time adjusted to find point at which there is
no white light saturation (for example, 10ms). Close shutter using software, not microscope.
Saturation reset to zero levels.
2.3) Exposure reset to 75% of zero saturation point, open shutter, capture image (Image
1), close shutter.
3) Procedure outlined in (2) is repeated for Telomere (Image 2) and Centromere (Image 3)
channels. Note: change channels using the filter wheel tool on the software, not the microscope.
4) Freehand draw around dapi image (Image 1), copy and paste into a new layer (ensure that the
new layer is also 12 bit before pasting cropped image in (Image 4).
5) With new dapi layer (Image 4) open go to density slice function and adjust setting till all of
dapi stained metaphase/nuclei is colored. Hit slice key, this generates a binary mask of the dapi
stain (Binary 1).
6) Open telomere image (Image 2) and go to the Boolean function option. Perform a Boolean
AND with the telomere image and the binary dapi mask (Binary 1). This will give a new image
of telomere spots that lie within the dapi binary mask (Image 5). Do the same for the centromere
image (Image 6).
7) Open the new telomere image (Image 5) and go to the density slice function. Adjust the
setting starting from zero and working upwards, a point will be reached where background pixels
will disappear, at this point slice image thereby generating a binary mask of the telomere images
(Binary 2). Do this for the centromere channel (Binary 3).
8) Select Image 5, go to measurements and select Binary 2 as a mask. Hit measure. This will
measure all of the individual telomere intensities and produce them as a numbered list. Export
the measurements as an excel file. Do the same for Image 6 using Binary 3. A number of
additional measurements are also generated such as Minimum intensity, Maximal Intensity,
Mean Intensity, Area.
9) Sum and divide all of the individual telomere intensities (TI) and all of the individual
centromere intensities (CI), such that (TI)/ (CI) = ratio of telomere to centromere.
10) Perform this analysis for as many nuclei are deemed appropriate to generate a data set