Promega DeadEndTM Fluorometric TUNEL System

Drugs to prepare in advance

(Advance preparation)
・ Put 1 ml of Proteinase K buffer in 10 mg of Proteinase K and dissolve. (Concentration of 10 mg /
ml)
→ Dispense and store at -20oC. This is diluted 500-fold with PBS before use.
・ DNase I buffer (stored at -20oC) Required amount 100 μl / sheet
・ RQ1 RNase-Free DNase I buffer (with DNase I of 5.5 to 10 units / ml) Required amount 100 μl /
sheet

Insulin-stained primary antibody; Guinea pig anti-insulin antibody (ab7842, Abcam) (1/100 diluted
with 1% BSA / PBS)

(Preparation for the second day)

Secondary antibody for insulin staining; Alexa Flour 546 goat anti-guinea pig IgG (A11074) (red)
(1/200 diluted with 1% BSA / PBS)
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(Protocol)

Deparaffinization
Lemosol I, room temperature, 5 minutes
Lemosol II, room temperature, 5 minutes

Hydration
100% ethanol I, room temperature, 5 minutes
100% ethanol II, room temperature, 3 minutes
95% ethanol, room temperature, 3 minutes
85% ethanol, room temperature, 3 minutes
70% ethanol, room temperature, 3 minutes
50% ethanol, room temperature, 3 minutes

Fixed

0.85% NaCl, room temperature, 5 minutes

PBS, room temperature, 5 minutes
4% PFA, room temperature, 15 minutes
PBS, room temperature, 5 minutes x 2 times

Penetration
Drain the water from the slide.
20 μg/ml Proteinase K solution 300 μl / sheet Place on slide, incubate at room temperature for 10
minutes.
PBS room temperature, 5 minutes

Fixed

4% PFA, room temperature, 5 minutes

PBS, room temperature, 5 minutes

Antibody reaction (p.5 IV A-5 ~)

(Positive control and antibody reaction of experimental sample are the same procedure)
(Negative control is omitted in ②, ③ is mixed with DW instead of rTdT Enzyme)
① Drop the solution on the slide.
② Equilibration Buffer 250 μl / sheet Room temperature, 10 minutes.
③ While waiting for 10 minutes, thaw the Nucleotide Mix on ice and adjust the rTdT incubation
buffer.

total amount of rTdT incubation buffer

= Equilibration Buffer 135 μl x number of samples + Nucleotide Mix 15 μl x number of samples +
rTdT Enzyme 3 μl x number of samples

④ After 10 minutes, dry the surrounding of equilibrated area with tissue paper.
⑤ Place the prepared rTdT incubation buffer 150 μl / sheet so as to cover the sample.
Cover with the included Plastic Cover slips to prevent it from drying out.
⑥ Wrap the wet chamber in aluminum foil and incubate at 37 ° C for 60 minutes in the dark.
⑦ 20 x SSC (10-fold diluted with DW) 300 μl / sheet 15 minutes at room temperature. (Stop
reaction)
⑧ PBS wash Room temperature, 5 minutes x 3 times
(In the case of TUNEL staining only, this is the end → Go to the encapsulation step.)

Blocking

3% BSA / PBS 350 μl / sheet Room temperature, 30 minutes

Insulin staining
Primary antibody: Guinea pig anti-insulin antibody (ab7842, Abcam)
1/100 diluted with 1% BSA / PBS 300 μl / sheet.
Over night at room temperature.
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(the next day)

PBS wash 5min x 3 times

Secondary antibody: Alexa Flour 546 goat anti-guinea pig IgG (A11074) (red)
1/200 diluted with 1% BSA / PBS 300 μl / sheet 60 minutes at room temperature.
PBS wash 5 min x 3 times
DW wash 10sec
Arrange one by one on the Kim towel in the drawer and dry. → Until it turns white.

Encapsulation

Specimen with Prolong Gold antifade reagent with DAPI and cover glass. Push the foam over the
cover glass.
Drop the nail polish top coat on the four corners and apply the cover glass to the edges.
Dry for about 10 minutes.

Observation

Observed with a fluorescence microscope

Fluorescein standard filter set
Observed with fluorescein green fluorescence at 520 ± 20 nm.
Observed with DAPI blue fluorescence 460 nm.
Insulin red fluorescence.
Storage

Slides can be stored overnight at 4 ° C under shading.