Cell material fixation and cryosectioning

3.3.1 Metaphase preparation from embryonic fibroblasts Material

• Colcemid-solution (10 mg colcemid/ml H2O bidest) in 1x PBS • Trypsin 0.05% (v/v), 0.02% EDTA (v/v) in 1x PBS

• Hypotonic solution: 0.075 M KCl

• Fixative: methanol/ glacial acetic acid 3:1 (v/v) • Ethanol (70%, 90%, 100%)

• 4mg/ml pepsin in 0.01N HCl • Heraeus Biofuge pico

• Certomat# _{R/H incubator}

In order prepare metaphases for cell cycle arrest at metaphase the spindle poison colcemid (10!l/ml) was added for 30min to a dividing, sub-confluent cell culture of mouse or chicken primary fibroblasts. First, cells were detached with trypsine and spinned down at 157g for 10min. After that, the supernatant was discarded, the pellet was resuspended in hypotonic buffer and incubated 15-20min at 37°C. Swelling of nuclei was stopped by adding 1ml of fixative. The cell suspension was then spinned (157g, 10min) down, resuspended in fixate, fixed for at least 25min at -20°C and cyclic washed 2-3x by spinning down and resuspension in fixative. The cell suspensions was stored at -20°C. Thereafter metaphase spreads from the cell suspension were prepared in a 55°C floating metal box according to (Deng et al. 2003) under standardized conditions. Next proteins were digested with pepsin solution (4mg/ml pepsin in 0.01N HCl for 1.5-2.5min at 37°C) and the metaphase spreads were incubated for 1h at 60°C to improve the FISH probe and the antibody accessibility. Last quality and quantity of metaphase spreads were checked under a phase contrast microscope.

3.3.2 Fixation of embryonic fibroblasts for 3D-FISH Material • 1x PBS (pH 7.4) • 2x SSC • 4% Paraformaldehyde in PBS 1x pH 7.4 • 0.5% Triton X-100 • 20% Glycerol in 1x PBS • Liquid nitrogen • 0.1N HCl • 2mg/ml pepsin in 0.01N HCl • 50% formamide/2x SSC (pH = 7.0)

To prepare fixed and 3-dimensionally preserved fibroblast nuclei for in situ hybridization experiments primary fibroblasts of mouse and chicken were grown on 26x76mm cover slips to 60-80% confluency in DMEM with 15% FCS. Thereafter, cells were fixed in 4% paraformaldehyde /1xPBS for 10min to maintain best possible the 3D-conformation of the nucleus. For 3D-Immuno-FISH fixed cells were stored in 1xPBS (see 3.6.6). Otherwise, permeabilization for normal 3D-FISH cell-fixations in five steps allow efficient FISH and antibody penetration: (1) treatment in 0.5% Triton X-100 in PBS for 20min plus 3x5min washes in 1x PBS; (2) 20% glycerol in PBS for; at least 1h (3) 4 freezing/thawing cycles in liquid nitrogen plus 3x5min washes in 1x PBS; (4) incubation in 0.1N HCl for 8min including 3x5 in 3x5min in 1x PBS (5) pepsinization (2mg/ml pepsin in 0.01N HCl at 37°C for 5-8min) followed by 2x5min 2x

SSC equilibration (Solovei et al. 2002). Finally, slides were stored in 50% formamide/2x SSC (pH = 7.0) at 4oC until 3D-FISH (see 3.6.5).

3.3.3 Fixation of embryos and adult tissue Material

• 1x PBS (pH 7.4, DPEC treated)

• Paraformaldehyde 4% in PBS 1x pH 7.4 • DEPC

• Test-Tube-Rotator 34528

Tissue was fixed in 4%PFA 1xPBS (DPEC) at 4°C over night in a rotation wheel to maintain the tissue integrity and nuclear 3-dimensional morphology. All solutions in contact with tissue for later RNA isolation were stirred over night with 0.1% active DPEC and subsequently autoclaved, to (di-ethyl-propyl carbonate) block RNAse activity by DEPC-binding to secondary and tertiary amines, hydroxy and thiol groups present in the enzymes catalytic domain and is pyrolyzed to ethanol and CO2 during

autoklaving. Next, fixed tissue was either processed for cryosections (3.3.4) or whole mount RNAish (3.5.2).

3.3.4 Cryoprotection and cryosectioning for chromogenic RNAish and 3D-FISH Material

• 1x PBS (pH 7.4, DPEC treated)

• Paraformaldehyde 4% in PBS 1x pH 7.4 • 0.1M phosphate buffer pH 7.4

• 5% sucrose in 0.1M phosphate buffer • 12.5% sucrose in 0.1M phosphate buffer • 20% sucrose in 0.1M phosphate buffer • Dry ice

• 95% ethanol • Razor blades • Test-Tube-Rotator

• Tissue freezing medium (JUNG, Oder Number 0201 08926) • Poly-A-Way Disposable Embedding molds (T-12)

• CM3000 Cryostat

• Super Frost#Plus slides

After fixation (see 3.3.3) tissue was cryoprotected in an increasing sucrose gradient in 0.1M phosphate buffer to prevent the tissue from drying and freezing injury by ice crystal formation. Then, tissue was washed 2x10min in 0.1M phosphate buffer,

equilibrated for 1h each in 5%, 12.5% at RT and finally over night in 20% sucrose in 0.1M phosphate buffer at 4°C on a rotation wheel. The day after the tissue was cut with a razor blade to fit the embedding mold dimensions. The embedding mold was filled with tissue freezing medium and tissue was orientated with a pipette tip. Chicken E21h, hindlimbs of mouse E13.0 and chicken E5.5, skin of mouse and chicken and mouse mammary gland were orientated horizontally. Mouse E7.0, head of mouse E13.0 and head of chicken E5.5 were orientated vertically. Finally, tissue blocks were frozen in a dry ice 95% ethanol bath (-78°C) and stored at -80°C until cryosectioning. 20!m cryosections were cut in a cryostat-microtome at -17°C, taken up with a room tempered Super Frost#Plus object slide and replaced to -80°C. 3.3.5 Freezing and cryosectioning for RNA FISH and qPCR

Material

• 1x PBS (pH 7.4, DPEC treated)

• Paraformaldehyde 4% in PBS 1x pH 7.4 • Liquid nitrogen

• Tissue freezing medium (JUNG, Oder Number 0201 08926) • Poly-A-Way Disposable Embedding molds (T-12),

• CM3000 Cryostat

• Super Frost#Plus slides

• 1mm PEN-membrane slides • 70%, 90%, 100% ethanol • Hematoxylin

• H20 (DEPC treated)

After dissection of mouse E13.0 the embryos were (see 3.2.2) placed in an embedding mold without fixation and immediately frozen to prevent RNA damage. Therefore embryos were quickly embedded in tissue freezing medium and snap frozen in liquid nitrogen (-196°C) to avoid RNA degradation and to prevent tissue damage from ice crystal formation, because snap freezing in liquid nitrogen below - 140°C produces vitreous ice with an amorphous structure. Tissue blocks were stored at -80°C until tissue sectioning in a cryostat-microtome at -17°C. After the uptake of cryosections, afore cut at 14!m with a room tempered sterile Super Frost#Plus object slide the cryosections were directly placed on dry-ice and subsequently fixed 15min in 4% PFA in 1xPBS (DPEC) in advance to RNA FISH (see 3.5.4). In contrast, 8!m cryosections for qPCR were mounted on sterilized (baked 3h at 180°C) 1mm PEN-membrane slides, then rapidly fixed 2.5min in ice cold 70% ethanol, briefly rinsed 3x in H2O (DPEC), stained 1min in hematoxylin, washed 1min in H2O (DPEC),

briefly dehydrated in a 70%, 90% and 100% ethanol series and finally placed on dry ice until the shortly consecutive laser microdissection (see 3.4.5).