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Home  |  Protocols  |  In situ hybridization and Immunochemistry  |  In Situ Hybridization on sections

Protocol - In Situ Hybridization on sections

For RNA detection on tissue sections

In Situ Hybridization on sections
Category:  In situ hybridization and Immunochemistry
Last revision: Apr 11, 2016
Contact
Name: Giovanna L. Liguori
Address: Institute of Genetics and Biophysics Via Pietro Castellino, 111 Naples 80131 ITALY
Email: giovanna.liguori@igb.cnr.it

 

Figure legend:

Part of Figure 2 from Olivieri et al., 2016 showing the expression of uc.8+ by in situ hybridization in A. normal bladder epithelium (NBE), B. low-grade BlCa tissues. A’, B’ represent enlargement of specific areas

Steps

Description Temperature Time Note
Put the slides in an apposite rack
Wash slides twice in Xylene
RT
5 minutes each
Check that paraffin disappears
Wash slides twice in 100% EtOH (after Xylene)
RT
5 minutes each
Sections become white
Wash slides in 75% EtOH/1X PBS
RT
5 minutes
Wash slides in 50% EtOH/1X PBS
RT
5 minutes
Wash slides in 25% EtOH/1X PBS
RT
5 minutes
Wash slides twice in 1X PBS
RT
5 minutes each
Place slides in 4% Paraformaldehyde/1X PBS
RT
10 minutes
Fixation
Wash in 1X PBT
RT
few seconds
Wash twice in 1X PBT
RT
5 minutes each
The samples are subsequently subjected to Proteinase K, which has the task of partially digest the tissue
RT
10 minutes
20µl PK (10 mg/ml) in 200 ml PBS
Wash twice in 1X PBT
RT
5 minutes each
Fix in 4% Paraformaldehyde/1X PBS
RT
10 minutes
Wash in 1X PBT
RT
few seconds
Wash twice in 1X PBT
RT
5 minutes each
Place slides in freshly prepared acetylation solution
RT
15 minutes
625 µl Acetic Anhydride in 250 ml 0.1 M TEA, shake well and use immediately
Wash in 1X PBT
RT
few seconds
Wash twice in 1X PBT
RT
5 minutes each
Add 100 µl of prewarmed (65°C) hybridization solution/slide. Place into cassette
65° C
for least 1 hour
Prehybridization
Prepare the probe: mix 200 ng/µl with 100 µl hybridization solution/slide an incubate
85° C
3 minutes
Add probe and cover slide with cover slip and incubate
65° C
over night
Hybridization
Remove cover slips by rising in 5X SSC
Post hybridization
Wash in 1X SSC/50% formamide
65° C
30 minutes
Wash in 1X TNE
37° C
10 minutes
Wash in 1X TNE/RNase A (20 µg/ml)
37° C
30 minutes
400 µl RNase A 10mg/ml in 200 ml TNE
Wash in 1X TNE
37° C
10 minutes
Wash in 2X SSC
65° C
20 minutes
Wash twice in 0.2X SSC
20 minutes each
Wash twice in 1X MABT
RT
5 minutes each
Block in 20% sheep serum/1X MABT (200 µl/slide) and incubate
RT
1 hour
Incubate with 2% sheep serum/1X MABT and anti-dig AP (1:2000 ratio)
4° C
over night
Incubate in humidified box
Wash in 1X MABT
RT
few seconds
Wash three times in 1X MABT
RT
5 minutes each
Wash in 1X NTMT
RT
10 minutes
Add NBT/BCIP and incubate
RT
1 hour - 3 days
at 4°C the reaction is slower
Wash in 1X NTMT
RT
few seconds
The stop reaction
Wash twice in 1X PBS
RT
5 minutes each
Dehydrate and mount the slides

Other informations

This protocol is suitable for 8-10 µm paraffin sections

 

Quality validation: Yes

Validation info

It has been validated by publication in peer reviewed journals

 

Citations
Olivieri M, Ferro M, Terreri S, Durso M, Romanelli A, Avitabile C, De Cobelli O, Messere A, Bruzzese D, Vannini I, Marinelli L, Novellino E, Zhang W, Incoronato M, Ilardi G1, Staibano S, Marra L, Franco R, Perdonà S, Terracciano D, Czerniak B, Liguori GL, Colonna V, Fabbri M, Febbraio F, Calin GA, Cimmino A. Long non-coding RNA containing ultraconserved genomic region 8 promotes bladder cancer tumorigenesis. Oncotarget. 2016; 1: 10:e 7833

 

Solutions

  • Hybridization solution

    Reagents F-concentration Quantity I-concentration
    Tris HCl pH 7.5
    10 mM
    2 M
    NaCl
    600 mM
    5 M
    EDTA
    1 mM
    0.5 M
    SDS
    0.25 %
    20%
    Dextransulfate
    10%
    100%
    Formamide
    50%
    100%
    Denhardt's
    1X
    100 X
    tRNA
    200 µg/ml
    10 mg/ml
    DNA (salmon sperm)
    100 µg/ml
    10 mg/ml
    H2O bd
    up to final volume

  • MAB 5X

    Reagents F-concentration Quantity I-concentration
    Acido Maleico
    23.3 g
    NaCl
    17.4 g
    H2O
    up to final volume

    Note

    Adjust pH to 7.5 with NaOH

    Only at pH 7.5 the solution dissolves

  • TNE 5X

    Reagents F-concentration Quantity I-concentration
    Tris pH 7.5
    50 mM
    2 M
    NaCl
    2.5 M
    5 M
    EDTA
    5 mM
    0.5 M

  • NTMT

    Reagents F-concentration Quantity I-concentration
    NaCl
    0.1 M
    5 M
    Tris HCl pH 9.5
    0.1 M
    1 M
    MgCl2
    0.05 M
    1 M
    Tween
    0.1 %
    100%
    H2O bd
    up to final volume

  • MABT

    Reagents F-concentration Quantity I-concentration
    MAB
    1X
    5X
    Tween
    0.1 %
    100%
    H2O bd
    up to final volume

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