Embed fresh-frozen tissues in OCT (optimal cutting temperature) compound and store them at -80°C until cryosectioning.

Section the tissues at 5-20 μm thickness and collect on SuperFrost Plus adhesion slides and store at -80°C until used for experiment.

When working with Drosophila tissues or other very fragile material, the tissues can be fixed for 20' in 4% PFA at room temperature, washed 4 times in PBS, cryoprotected in 30% sucrose in PBS overnight, and finally embedded in OCT. This slightly reduces the detection efficiency but keeps a much better tissue morphology. Drosophila tissues or other very fragile material, the tissues can be fixed for 20' in 4% PFA at room temperature, washed 4 times in PBS, cryoprotected in 30% sucrose in PBS overnight, and finally embedded in OCT. This slightly reduces the detection efficiency but keeps a much better tissue morphology.

Take the slides from -80°C and leave them at room temperature (RT) for 3-5 min to air dry and reach room temperature.

Fix with freshly prepared 3% formaldehyde in PBS for 5 min at RT.

Formaldehyde solution can be applied directly on top of the section, or the slide can be submerged in a large amount of fixative.

Discard formaldehyde from tissue section and wash with PBS at RT 3 times

Permeabilize the tissue with 0.1 M HCl (in H2O) at RT for 5 min.

This can either be done by submerging the slide in HCl or by covering the section as in step 3

Permeabilization of tissue needs to be optimized. Additional pre-treatments are possible such as incubation with Pepsin. Permeabilization should be optimized depending on the source of the tissue. In many of the cases this doesn't seem a crucial step.

After permeabilisation, wash the sections with PBS two times.

Dehydrate the sections in order to aid the adhesion of SecureSeal chambers.

Submerge sections/slides in 70% Ethanol for 1min.

Transfer and submerge in ~100% Ethanol for 1min.

Remove slide and let air dry.

Note that it is also possible to mount SecureSeal chambers without dehydrating slides, you just have to ensure a dry surface area around tissue for proper adhesion of the chamber.

Apply SecureSeal hybridization chambers of the appropriate size over the tissue.

Wash the section and the chamber by applying PBS-T to chamber inlet and then wash once with PBS. Let PBS remain in chamber until the reagents for the next steps are prepared.

The PBS-T will coat with detergent the surface of the tissue, slide and chamber, making the next steps of washing and pipetting much easier.

Apply and remove solutions to chamber by tilting the slide/chamber slightly and pipetting into lower inlet. This will aid in preventing bubble formation within the chamber.

Prepare the following hybridisation mix.

A	B	C	D
Reagent	Stock [ ]	Final [ ]	Amount
DEPC H2O			39.5
20x SSC	20x	2x	5
Formamide	100%	10%	5
Chimeric PLPs	1 uM each	10 nm each	0.5
Total			50

Remove the PBS from the chamber, add the hybridisation mix and incubate at 37 degrees C overnight

Remove the hybridisation mix.

Wash twice in 10% formamide in 2xSSC.

Wash twice in 2xSSC.

Prepare the following ligation mix.

A	B	C	D
	stock [ ]	final [ ]
DEPC h2O			27,25
Hm T4 Rnl2 buffer (No Mg)	10x	1x	5
MgCl2	25mM	2mM	4
ATP 1 mM	1mM	100 uM	5
RCA primer	1µM	0.05µM	2,5
RiboProtect (RNase inhibitor)	40U/µl	1U/µl	1,25
T4 Rnl2 (homemade)	10U/µl	0,5U/µl	2,5
total			50

Remove the 2xSSC and add the ligation mix to the chamber.

Incubate for 2 hours at 37 degrees C.

Wash twice with PBS.

Prepare the following amplification mix.

A	B	C	D
Reagents
DEPC H20			34
phi29 buffer	10X	1X	5
glycerol (Placed at RT)	50%	5%	5
dNTPs	25 mM	0.25mM	0,5
BSA	20 ug/ul	0.2µg/ml	0,5
phi29 polymerase	10 U/ul	1U/µl	5
total			50

Incubate at 30 degree celsius O/N

After overnight incubation, remove reagents from chamber.

Wash chamber twice with PBS. Let PBS remain until next step.