Rastreo de dificultades a partir de la observación directa

In situ hybridization was carried out on cryostate sections of 20µm thickness or vibratome sections of 60 µm thickness whith the latter giving better results probably due to the thickness that shows stronger signal and better penetration of the in situ probe from both sides. In general the in vitro transcription and the in situ hybridization were carried out under semi-sterile conditions using sterile pipet tips and gloves.

An in situ hybridization can be divided into two main steps: (i) generating the anti- sense RNA specific for the according RNA in the tissue by in vitro transcription (described in chapter 6.8.1 In vitro transcription); (ii) hybridization of the RNA probe and its detection in the section (described in 6.8.2 In situ hybridization). In general the in situ hybridizations were developed in a stain visible in normal light. However, in special cases a fluorescent in situ hybridization protocol was applied (see chapter 6.8.3).

6.8.1 In vitro transcription: generation of digoxigenin labelled probes

The cDNA containing ISH plasmid was linearized by use of an enzyme at the end cDNA probe insert. Therefore, the RNA polymerase falls off the DNA strand at a defined point after transcribing the according cDNA into RNA.

Linearization of 1 µg DNA was performed with 1 µl of the according enzyme and 2 µl 10 x buffer in 20 µl final volume. After digestion DNA was purified by use of a column (Qiagen) or Phenol extraction (described above).

Transcription was performed in an 1.5 ml reaction tube by using 1 µg DNA, 2 µl digoxigenin labelled dNTPs (Roche), 4 µl 5 x buffer (Stratagene), 1 µl RNAse inhibitor

6 In situ hybridization 143

(Roche) and 1 µl of the according RNA Polymerase (T3, T7, Sp6) (Stratagene). The mixture was kept for 1 - 2 hour at 37°C and RNA was cleaned over a RNA binding column (Qiagen). 1 µl of the resulting RNA probe was examined on a gel for RNA presence. All probes were then tested on sections. As a rule of thumb 2 µl per 100 µl hybridization buffer was used first until the optimal concentrations were determined.

6.8.2 In situ hybridization

For vibratome sections the hybridization was performed in a sterile Eppendorf tube and the following steps in 24- or 6-well plates. For cryostate sections the hybridization was carried out in chambers containing chamber solution (1 x SSC containing 50 % Formamide) to avoid drying of the sections overnight. The amount of hybridization buffer was calculated per slide or sections: For a cryostate section 150 µl hybridization buffer was used or alternatively 500 µl for 3 - 4 vibratome sections in one reaction tube. After adding the probe to the hybrization buffer the mixture was heated up to 74°C for 4 minutes to separate RNA strands and mixed again. Vibratome sections were added to the tube containing the hybrdisation solution or the buffer was put onto the slides with the cryostate sections and sterile glass slides (shortly breamed) were carefully put on top and the chamber solution was added into the chamber. Hybrization was carried out in an oven or water bath at 65°C overnight.

Hybridization buffer 10 ml Formamid 5 ml 20 x SSC 1.5 ml Deinhardt’s solution 50x 200 µl 50 % Dextran sulfate 2 ml t-RNA 500 µl H2O Up to 10 ml

The next day the washing solution was pre-warmed in a water bath to 65°C. Cryostate sections were moved to a sterile washing chamber, vibratome sections were moved into a 6-well plate containing washing solution. Sections were incubated twice for 30 minutes at 65°C in washing solution followed by two washing steps in 1 x MABT for 30 minutes at room termperature.

Washing solution 50 ml 20 x SSC 2.5 ml 10 % Tween 0.5 ml Formamide 25 ml H2O 22 ml MABT 5x 2 l

Maleic acid (500 mM end concentration) 116.08 g NaCl (750 mM end concentration) 87.7 g

10% Tween 20 20 ml

H2O 1980 ml

Adjust pH to 7.5 ~ 70 g NaOH

Blocking solution was prepared. For vibratome sections 500 µl per probe was use in a 24-well plate and for cryostate sections 500 µl per slide was used. The blocking solution was added drop wise onto the slides that were put back into the chamber and covered by parafilm to avoid drying. Vibratome sections were transferred from a 6-well to a 24-well plate and sections were incubated for 1 hour at room temperature. The coupled to alkaline phosphatase (Fab Fragments, Roche, 1:2000) was diluted in blocking solution meanwhile. The blocking solution on the slides was discarded and replaced by antibody containg solution and covered again by parafilm. The vibratome sections were transferred to the next well containing the antibody solution. Sections were incubated at 4°C over night.

Blocking solution 10 ml

MABT 5 x 2 ml

Bovine Serum 2 ml

10 % Blocking Reagent 2 ml

H2O 4 ml

The next day, sections were washed 4 – 5 times in 1 x MABT buffer and twice in freshly prepared AP staining buffer. NBT and BCIP (3.5 µl per ml AP staining buffer) (Roche) were added to the AP buffer and put onto the sections until the desired staining intensity was reached. Reaction was stopped by washing the section in autoclaved water. Sections were processed for immunohistochemistry as described above or mounted and coversliped.

6 In situ hybridization 145 AP staining buffer 50 ml 1 M Tris pH 9.5 5 ml 10 % Tween 20 0.5 ml 5 M NaCl 1 ml 1 M MgCl2 2.5 ml H2O Up to 50 ml

6.8.3 Fluorescent in situ hybridization

Fluorescent ISHs were performed for combination with immunohistochemistry and colocalisation at the confocal level. The tyramide Kit (Perkin Elmer) was applied in combination with anti-Digoxigenin antibody coupled with horse-radish-peroxidase (anit-DIG-POD, Roche, 11 207 733 910).

Prehybridization

- Cut sections at 75 μm (ISH immediately after cutting is best, storage at 4°C in 4% PFA is possible)

- Remove sections for use and wash in PBS (make with DEPC water) 2 X 5 min - Wash briefly (30 seconds) in DEPC water

- Incubate for 5 min in 2.3 % sodium metaperiodate (dissolve in DEPC water) - Wash briefly (30 seconds) in DEPC water

- Wash in 100 mM Tris pH 7.5 for 5 min

- Incubate in 1 % sodium borohydrate (dissolved in 100 mM Tris pH 7.5) for 10 min - Wash briefly in PBS

- Wash 2 X 5 min in PBS - Incubate 5 min in 4% PFA - Wash 2 X 5 min in PBS

- Incubate in pre-hybridization solution for 1 hour at 55°C

- Incubate in hybridization mix with specific probes overnight at 55°C

Postbridization

- Wash 2 X 30 min in 2X SSC, 50 % formamide at 55°C - Wash 5 min in 2 X SSC at 55°C

- Wash 30 min in 0.1 X SSC, 60°C - Wash 2 X 5 min in Buffer 1

- Block for 30 min at room temperature in TNB blocking buffer

- For DIG labeled probes, incubate overnight at 4°C with anti-digoxigenin-HRP (anti- DIG-POD) diluted 1:100 - 1:250 in TNB buffer

- Wash 3 X 5 min in TNT buffer

- Incubate in amplification diluent solution (provided with the Kit) for 10 min - Wash 3 x 5 min in TNT buffer

- For fluorescence visualization, incubate in Streptavidin conjugated fluorophore (Texas Red or Rhodamine; NEL721) diluted 1:100 – 1:500 in TNB buffer

- Wash 6 X 10 min in TNT buffer or overnight - Mount and coverslip

Reagents

NTE: Prehybridization Solution:

NaCl 5 M, 50 ml 25 ml Formamide

Tris pH 8 1M, 5 ml 7.5 ml SSC 20X

EDTA 0.5M pH8, 1 ml Water to 50 ml

Water to 500 ml final volume

Buffer 1 (10 X, 1 l):

Hybridization Mix (10 ml): 60.55 g Trisbase (FW= 121.1g/mol)

50 x Dendhardt’s solution 200 μl 87.66 g NaCl (FW = 58.44 g/mol)

t-RNA 500 μl Volume to 1 L, pH 7.5

50 % Dextran sulfate 2 ml

Formamide 5 ml TNT Wash Buffer:

SSC 20 X 1.5 ml 0.1 M Tris, pH 7.5

Water 800 μl 0.15 M NaCl

0.3% Triton X-100

TNB Buffer: 0.1 M Tris, pH 7.5

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0.15 M NaCl

0.5 % (wt/vol) blocking reagent (TSA kit, Perkin Elmer)

Stir for 1-2 hr at 60°C until fully dissolved. Filter and store at <20 °C. Solution should be milky white in colour with no particulates.