Protocols

Y-Chromosome FISH Protocols

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Unless noted otherwise, all incubations take place at room temperature
1. Equilibrate frozen sections to RT
2. Wash in 2xSSC, incubate two times, 3 min each incubation (2x3min)
3. Incubate in 0.2 M HCL for 10 min at room temperature
4. Wash in 2xSSC 2x3min
5. Incubate in 1M NaSCN for 20 min at 75-80°C
6. Wash in 2xSSC 2x3min
7. Water rinse
8. Ethanol dehydrate (70%, 95%, 100%), 1 min each
9. Air-dry slides
10. Hybridization:pre-heat probe to 75°C, vortex to mix
a. Add 10-12 µl preannealed probe to slides and cover with a small coverslip (18mmx18mm).
b. Seal with rubber cement - must be certain that edges of coverslip are sealed completely (can't use too much!)
c. Incubate @ 75°C for 5 min.
d. Incubate @ 37°C overnight in moist chamber
11. Carefully remove rubber cement and coverslip, wash in 2xSSC 2 min
12. Wash with 0.4x SSC/0.3%NP40 at 55 C for 3 min.
13. Wash 2xSSC 2 min
14. Block:incubate in 4xSSC/3%BSA/0.1%Tween20 for 30 min at 37°C
15. Detection:dilute anti DIG-rhodamine (Roche cat# 1 207 750) 1:20 in 4xSSC/1%BSA/0.1%Tween20 and incubate at 37°C for 45 min.
16. Wash well with 2xSSC
17. Rinse slides in water
18. Air-dry slides in the dark
19. Add a drop of vectashield mounting medium with DAPI (Vector cat# H-1200), coverslip and view.
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Unless noted otherwise, all incubations take place at room temperature
1. Heat tissues to 60°C for 10 mins
2. Deparaffinize and rehydrate tissue sections through series of xylenes and ethanol incubations
a. Xylenes or alternative (Citrisolve, Histoclear) incubate 2 times, 5 min each incubation (2 x 5 min)
b. 100% ethanol, 5 min
c. 95% ethanol, 5 min
d. 70% ethanol, 5 min
3. Rinse the slides once in dH20
4. Wash with PBS for 5 mins
5. Antigen retrieval:Preheat BD retrievagen A solution (BD Pharmigen cat# 550524) in coplin jar in steamer. Add slides to coplin jar and incubate for 10-20 min in steamer then let stand at room temperature for 20 min.
6. Wash 2xSSC 2x3min
7. Incubate in 0.2 M HCL for 12 min at room temperature
8. Wash in 2xSSC 2x3min
9. Incubate in 1M NaSCN for 20 min at 75-80°C
10. Wash in 2xSSC 2x3min
11. Water rinse
12. Ethanol dehydrate (70%, 95%, 100%), 1 min each
13. Air-dry slides
14. Hybridization:pre-heat probe to 75°C, vortex to mix
a. Add 10-12µl preannealed probe to slides and cover with a small coverslip (18mmx18mm).
b. Seal with rubber cement - must be certain that edges of coverslip are sealed completely (can't use too much!)
c. Incubate @ 75°C for 5 min.
d. Incubate @ 37°C overnight in moist chamber
15. Carefully remove rubber cement and coverslip, wash in 2xSSC 2 min
16. Wash with 0.4x SSC/0.3%NP40 at 55°C for 3 min.
17. Wash 2xSSC 2 min
18. Block:incubate in 4xSSC/3%BSA/0.1%Tween20 for 30 min at 37°C
19. Detection:dilute anti DIG-rhodamine (Roche cat# 1 207 750) 1:20 in 4xSSC/1%BSA/0.1%Tween20 and incubate at 37°C for 45 min.
20. Wash well with 2xSSC
21. Rinse slides in water
22. Air-dry slides in the dark
23. Add a drop of vectashield mounting medium with DAPI (Vector cat# H-1200), coverslip and view.
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Blocking Solution

For 100 mls:
3g BSA - end conc. 3% BSA
20mls 20x SSC - end conc. 4x SSC
100µl Tween20 - 0.1% Tween in 100mls
H2O up to 100 mls

Detection Solution

For 100 mls:
1g BSA - end conc. 1% BSA
4xSSC/0.1% Tween up to 100 mls

Hybridization Mixture

For 20 mls:
8 mls 50% Dextran Sulfate - end conc. 20% Dextran Sulfate
4 mls 20x SSC - end conc. 4x SSC
8 mls H2O
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Generate Y chromosome template DNA
If you are starting with an aliquot of 2-4 µl template DNA, amplify the DNA several (2-3)
times by DOP-PCR to generate Y chromosome template. This will be your Y chromosome
template stock. Then each time you make probe use DNA from this stock for another batch
(8 reaction) DOP-PCR and subsequent labeling. Do not repeatedly amplify DOP-PCR as this
can bias the oligos generated by selecting for a subpopulation of oligos that amplify
particularly well.


We usually generate Y probe in bulk by making a master mix for 10 PCR reactions.
We then make 8 DOP-PCR reactions, have one no DNA control and 1 excess reaction
for pipetting error. This bulk reaction gives us 1.6 mls of Y probe, roughly enough
to stain 160 slides.

DOP-PCR (degenerate oligonucleo primed-polymerase chain reaction)

PCR reaction, 50 µl total volume

1 reaction10 reactions
ADD:
Water40.3 µl403 µl
10x PCR buffer5 µl50 µl
10 mM dNTP1.2 µl12 µl
100 µM 6AI primer1 µl10 µl
Taq stock 5U/ µl0.5 µl5 µl
(Roche cat# 1 435 094)

Take out 48 µl for no DNA control, place in a labeled 0.2ml tube

1 reaction9 reactions
ADD:
Mouse Y chromosome DNA2 µl18 µl

Pipette 50 µl into 8 tubes


6AI Primer= CCG ACT CGA GNN NNN NTA CAC C
where N is an equal concentration of all nucleotides

Cycle conditions
PCR cycledenaturingannealingextension
1-2 cycles45sec/94°C45sec/15°C12min/37°C
5 cycles40sec/94°C45sec/37°C4min/66°C
24 cycles40sec/94°C45sec/54°C4min/66°C

For bulk DOP-PCR, pipet all the reactions together after cycling. Then run 5 µl pooled
DOP-PCR reaction and 5 µl no-DNA control on a 2% agarose gel alongside a 100 bp marker.
The DOP-PCR reaction produces a smear that runs from approximately 300 to 2.5 kb.
Next ethanol precipitate the pooled DOP-PCR DNA overnight by adding 1/10 the volume of
3M NaOAc, vortexing and then adding 2.5x the volume 100% ethanol and leaving at -20°C ON.
The next day centrifuge the solution at 14,000 rpm for 30 min to pellet the DNA. Next wash
the pellet in 70% ethanol and air-dry. Resuspend the pellet in the starting volume of distilled
water (50 µl per PCR reaction, 400 µl for 8 reactions pooled).

Making the Y probe
DIG-Nick Translation
The next step involves digesting the Y chromosome template DNA (DOP-PCR reaction)
into smaller pieces and labeling these pieces with digoxigenin. This is done using the DIG-Nick
translation kit (ROCHE cat# 1745876). Take out 5-20 µl of newly resuspended DOP-PCR
and set aside.
1 reaction8 reactions
ADD:
Y Chromosome Template (DOP-PCR)approx 50 µlapprox 400 µl
DIG-Nick Kit mix12.5 µl100 µl

This is now your DIG-Nick reaction solution. Incubate at 15 °C for 90 min to 2 hrs. Put the
reaction on ice and run the reaction on a 2% agarose gel alongside the 5 µl of DOP-PCR
you set aside earlier and some 100bp marker. You expect to see smear of fragments ranging
from 150-500 bp. If this is the case, stop the reaction. If the smear still contains larger sized
fragments, return the reaction to 15°C and check again on a 2% agarose gel every 10-20 min
until the reaction contains the appropriate fragment sizes.


To stop the reaction:

1 reaction8 reactions
ADD:
DIG-Nick reaction62.5 µl500 µl
0.5M EDTA4 µl32 µl

and incubate at 65°C for 10 min.

Ethanol precipitation with carrier and blocker DNA
Aliquot the DIG-Nick reaction by placing 100 µl of it into several 1.5 ml eppendorfs. Scale up
or down as necessary.

ADD:
DIG-Nick reaction100µl
Sonicated Salmon sperm 10mg/ml
(Stratagene cat#21190)
60µl

Cot-1 DNA 1mg/ml
(Invitrogen cat#18440-016)

100 µl
3 M NaOAC (then vortex)26 µl
100% ethanol800 µl

Vortex again and leave ON at –20°C. Centrifuge at 14,000 rpm for 30 min to pellet the DNA.
Next wash the pellet in 70% ethanol and air-dry. Resuspend the pellet in 100 µl formamide DI
(deionized). Make sure the pellet is completely resuspended by heating the solution to 42°C
for 10 min or higher if necessary. Then add an equal volume of hybridization buffer and store
the probe at -20°C.


Hybridization Buffer
For 20 mls:
8 mls 50% Dextran Sulfate - end conc. 20% Dextran Sulfate
4 mls 20x SSC - end conc. 4x SSC
8 mls H2O
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  1. Take about 100 ul blood from mouse into heparanized tube
  2. Dilute blood in 5 ml PBS in a 15 ml conical.
  3. Spin tubes for 7 min at 1000 rpm
  4. Remove supernatant
  5. Gently resuspend cells in remaining volume by flicking tube with finger
  6. Slowly and gently resuspend the cells in 5 mls of 0.075 M KCl, 37oC.
  7. Incubate 5 minutes at 37oC to allow cells to swell
  8. Centrifuge 1000 rpm x 7 minutes
  9. Remove KCl leaving a few drops of the solution and resuspend by tapping with finger
  10. Dropwise with constant mixing, add 5 mls cold freshly prepared fixative (3:1 Methanol:Glacial Acetic Acid)
  11. Vortex for 30 seconds at lowest speed setting
  12. Incubate on ice for 30 minutes
  13. Centrifuge and remove supernatant
  14. Add 5 mls fresh ice-cold fixative
  15. Perform steps 13 and 14 two more times (3 washes total after 30 min incubation)
  16. The final volume of fixative will depend on the cell pellet, and spreads on the slide will need to be checked for cell density
  17. Drop 10-20 ul of cell suspension on a clean microscope slide (high quality precleaned slides, soak in 100% EtOH overnight, wash with distilled H2O and wipe clean with lint free paper towel), drop from height of about 6 inches
  18. Dehydrate the slides through an EtOH series (70%, 90%, and 100% - 5 minutes each), and air dry.
  19. Store slides in box covered with parafilm at -20C
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Immunohistochemistry Protocols

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Antibody -Dako mouse monoclonal clone Bu20, cat# M0744

  1. Heat tissues to 60°C for 10 mins
  2. Deparaffinize and rehydrate tissue sections through series of xylenes and ethanol incubations
    1. Xylenes or alternative (Citrisolve, Histoclear) incubate 2 times, 5 min each incubation (2 x 5 min)
    2. 100% ethanol, 5 min
    3. 95% ethanol, 5 min
    4. 70% ethanol, 5 min
  3. Rinse the slides once in dH20
  4. Wash with PBS for 5 mins
  5. Antigen retrieval: Preheat BD retrievagen A solution (BD Pharmigen cat# 550524) in coplin jar in steamer. Add slides to coplin jar and incubate for 10 min in steamer then let stand at room temperature for 20 min.
  6. Wash PBS 2 x 5 min
  7. Antigen retrieval: warm Proteinase K (200µg/ml ) solution to 37°C. Incubate slides for 1 min at RT with proteinase K solution.
  8. Block: Quench endogenous peroxidase using 3% H202 in methanol for 20 mins @RT
  9. Block: incubate in PBS/5% BSA for 15 min at RT.
  10. Primary antibody: Dilute anti-BRDU mouse monoclonal 1:100 in PBS/2.5% goat serum,/0.05% Tween20 overnight at 4°C.
  11. Wash in PBS 3 x 5 min
  12. Block: biotin block using Vector kit (cat# SP-2001)
  13. Wash PBS 2 x 5 min
  14. Secondary antibody: dilute anti-mouse biotin (Molecular Probes B-2763) 1:100 in PBS and incubate for 1HR at room temperature
  15. Make ABC solution (Vector Vectactain ABC Elite PK-6100)
  16. Wash PBS 2 x 5 min
  17. Incubate in ABC solution for 20 min at room temperature
  18. Wash PBS 2 x 5 min
  19. Develop: use DAB or other peroxidase substrate
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GFP Antibody - Clontech mouse monoclonal, clone JL-8 cat# 8371-2

Prepare MOM solutions according to manufacturers instructions- Vector MOM Peroxidase kit (cat# PK-2200) or MOM basic kit (cat# BMK-2202). All incubations take place at room temperature unless indicated.

  1. Heat tissues to 60°C for 10 mins
  2. Deparaffinize and rehydrate tissue sections through series of xylenes and ethanol incubations
    1. Xylenes or alternative (Citrisolve, Histoclear) incubate 2 times, 5 min each incubation (2 x 5 min)
    2. 100% ethanol, 5 min
    3. 95% ethanol, 5 min
    4. 70% ethanol, 5 min
  3. Rinse the slides once in dH20
  4. Wash with PBS for 5 mins
  5. Antigen Retrieval: Preheat BD retrievagen A solution (BD Pharmigen cat# 550524) in coplin jar in steamer. Add slides to coplin jar and incubate for 20 min in steamer then let stand at room temperature for 30 min.
  6. Wash with PBS 2 x 5 min
  7. Block: Incubate slides in PBS/3% BSA/0.05%Tween20 for 30 min at 37°C.
  8. Wash in PBS 2 x 5 min
  9. Primary Antibody: Dilute anti-CD45 1:20 (Santa Cruz cat# sc-18846) or isotype in MOM diluent, incubate for 1 HR at RT
  10. Wash PBS 2 x 5 min
  11. Secondary Antibody: anti-rat-alexa 647 (Mol Probes) 1:100 in PBS and incubate for 1 HR at RT
  12. Wash PBS 2 x 5 min
  13. Block: biotin block using Vector kit (cat# SP-2001)
  14. Wash PBS 2 x 5 min
  15. Block: MOM block, 30 min at 37°C
  16. Wash PBS 2 x 5 min
  17. Diluent, tip off
  18. Primary Antibody: Dilute anti-GFP 1:250 or isotype in MOM diluent and incubate for 1HR at RT
  19. Wash PBS 2 x 5 min
  20. Secondary Antibody: Incubate in MOM biotinylated reagent for 10 min at RT
  21. Wash PBS 2 x 5 min
  22. Tertiary Antibody: Dilute Streptavidin-alexa 488 (Mol Probes) 1:500 in PBS/1%BSA and incubate for 30 min at 37°C
  23. Wash PBS 2 x 5 min
  24. Antigen Retrieval: 0.5% 1x trypsin (Gibco cat#16438, 0.25% trypsin, 1mM EDTA) preheated to 37°C, incubate 30-35 seconds
  25. Wash PBS 2 x 5 min
  26. Primary Antibody: Dilute anti-pankeratin (DAKO cat#Z0622) 1:500 in MOM diluent incubate ON at 4°C
  27. Wash PBS 2 x 5 min
  28. Secondary Antibody: Dilute anti-rabbit-alexa 568 (Mol Probes) 1:100 in PBS/1%BSA and incubate for 30 min at 37°C
  29. Wash and view
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GFP Antibody-Molecular probes rabbit polyclonal, cat# A11122
*Note: works best on lung, variable on other tissues

  1. Heat tissues to 60°C for 10 mins
  2. Deparaffinize and rehydrate tissue sections through series of xylenes and ethanol incubations
    1. Xylenes or alternative (Citrisolve, Histoclear) incubate 2 times, 5 min each incubation (2 x 5 min)
    2. 100% ethanol, 5 min
    3. 5% ethanol, 5 min
    4. 70% ethanol, 5 min
  3. Rinse the slides once in dH20
  4. Wash with PBS for 5 mins
  5. Block: Quench endogenous peroxidase using 3% H202 in methanol for 20 mins @RT
  6. Wash PBS 2 x 5 min
  7. Block: incubate in PBS/1%BSA for 20 mins at 37°C
  8. Wash PBS 2 x 5 min
  9. Primary Antibody: dilute anti-GFP (Mol Probes cat# A11122) or isotype control 1:100 in PBS/1%BSA, incubate 1 hour at 37°C
  10. Wash PBS 3 x 5 min
  11. Secondary Antibody: dilute anti-rabbit HRP (Dako cat#PO448) 1:100 in PBS/1%BSA and incubate for 30 min at 37°C
  12. Wash PBS 5 x 5 min
  13. Develop: use DAB or other peroxidase substrate
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