H ISTOCHEMISTRY AND I MMUNOHISTOCHEMISTRY

PAS Staining

Paraffin sections of implantation sites were incubated twice for 10min in xylene followed by rehydration in a series of 100%, 96% and 70% ethanol for three minutes each.

113 After washing in distilled H2O, the sections were incubated in Periodic acid 0.5% for 5 min.

Sections were washed through a series of 3 fast changes of distilled H2O and incubated in Schiff’s Reagent (0.5% Basic fuchsin; 0.5% Sodium metabisulfite; 0.2% Activated charcoal, filtrated) for 20min.

Sections were rinsed 7min under running tap water, stained 1min in Hematoxylene GILL-3 and rinsed again 7min under running tap water. Dehydration in an ascending ethanol series was followed by xylene incubation for clearance and mounting.

Nuclear Fast Red Counterstaining

Subsequent to β-galactosidase staining or to DAB-substrate to visualize HRP-coupled antibodies, the slides were washed in PBS for 15min at room temperature, rinsed in distilled water and counterstained with Nuclear Fast Red (Vector) for 5min. Sections were washed in distilled H2O for 10min and dehydrated through a ethanol series (twice 1min each in 70%, 95% and 100%ethanol). Sections were cleared in xylene twice for 5 to 10 minutes and mounted in xylene based medium.

β−galactosidase Staining

Expression of the lacZ reporter gene present in the replacement vectors for the Sall1, Sall2 and Sall3 genes was visualized by staining whole embryos and frozen tissue sections for β-galactosidase activity. A β-galactosidase staining solution containing Bluo-Gal substrate (20mM K3; 20mM K4; 0.01% Na-deoxycholate; 2mM MgCl2; 0.02% (v/v) NP-40; 1mg/ml Bluo-Gal) was used. Staining was carried out at 37°C over night.

Thionine Staining

Staining solution was prepared by slow addition of 0.625g Thionin powder to a solution of 191ml water, 9ml NaOH-1M, 50ml acetic acid-1M at 60°C. Solution was boiled for 45 minutes, filtrated and stored at 4°C for a maximum of 4 months. Staining was done subsequent to incubation in water and takes 5 minutes. Sections were dehydrated in an ethanol series directly after and mounted in xylene based medium.

Immunofluorescence

Imunofluorescent stainings were performed on 18 μm cryostat sections prepared as described. Sections were rinsed for 10 minutes in PBS. Blastocysts were fixed 5 minutes in

2% paraformaldehyde in PBS, permeabilized in 0.2% Triton X100 for 5 minutes and directly placed on a coated glass slide and allowed to dry in. For blocking, the tissue sections were incubated for 2 hours with 5 % (v/v) 2^nd antibody species serum in TBS/0.4 % Triton X-100 at room temperature. sections were washed twice with TBS/0.4% triton X-100 and incubated with the primary antibody at the appropriate dilution in TBS/0.4% triton X-100 O/N at 4°C.

The slides were rinsed twice with TBS/0.4% triton X-100 and incubated with fluorescent-labelled secondary antibody in TBS/0.4 % Triton X-100 for 1 hour at room temperature in the dark. The slides were washed twice in 1xTBS/0.4% triton X-100, counterstained in 1xDAPI for 5 minutes and mounted with 50μl of slow fade reagent (Prolong Antifade kit, Molecular Probes) according to the manufacturer’s indications. Slides were stored at 4°C in the dark.

Immunohistochemistry

Immunohistochemical stainings were performed on sections fixed in 4%

paraformaldehyde in PBS for 2-12 hours. Paraffin sections were initially incubated in xylene for 3x5 minutes to dissolve the paraffin followed by rehydration in a series of 100%, 95% and 70% ethanol (3 minutes each). After washing in PBS for 5 minutes, antigen was unmasked by boiling 1 hour in unmasking solution (Vector) and endogenous peroxidases were inactivated by incubation for 10 minutes in 3 % H2O2 in PBS followed by 5 minutes washing in PBS with agitation if DAB stainings were performed.

The sections were incubated with blocking buffer (0.2 % Triton, 1 % Glycine, 3% BSA, 5 % normal serum in PBS) in a wet chamber for 2 hours at room temperature. After blocking, the sections were incubated with primary antibody in blocking buffer in a wet chamber ON at 4°C. The slides were washed for 3x20 minutes in wash buffer (0.4 % Triton, 1 % Glycine, 3

% BSA in 1xTBS) with agitation. The sections were incubated with biotinylated secondary antibody (vector) in blocking buffer in a wet chamber for 1 hour at room temperature and then washed as after primary antibody. The staining procedure using the ABC reagent and DAB substrate was performed according to the manufacturer’s recommendations. The sections were counterstained with methyl green stain (1:8 dilution from 2 % stock 10 minutes) dehydrated 2x10 minutes in 1-Butanol, cleared in xylene and mounted with 60 μl Eukitt.

Embryo Staining for Cartilage and Bone

In order to reveal the skeletal structure and cartilage formation at e18.5, the following protocol was performed. Mice were sacrificed and placed in tap water for 24 hours. The skin was carefully peeled off with forceps by making a cut around the waist, neck, and base of the

115 limbs and pulling the skin back intact. Mice were eviscerated and fixed in 95%ethanol for 3-5 days. Staining for cartilage was performed by placing the mice in Alcian Blue solution (0.015% w/v) (Sigma). Staining was performed for 24 hours. The mice were then rinsed twice in 95% ethanol and then let sit again in 95%ethanol for 24 hours. The mice were then placed in 2% KOH for a period of time that allows them to be cleared. Counterstaining for bone with Alizarin Red solution (0.005% w/v) (Sigma) stain was performed for 1-3 hours until an optimal level of staining was observed. The mice were cleared by placing them in 2% KOH of decreasing strengths. Initially a 2% KOH solution was used until the mice were almost clear.

The clearing process was completed in the following ratios of 2% KOH to glycerol; 80:20, 60:40, 40:60, 20:80. The mice were stored in 2% KOH, glycerol (20:80).