Anti digoxigenin alkaline phosphatase fab fragments

In situ hybridization was performed as described previously^{43 (link)}. Briefly, riboprobes for collagen X and for Osteocalcin⁴⁴ were generated by PCR using mouse growth plate cDNA as template and primers that contained an SP6 promoter. Single-stranded digoxigenin-labeled riboprobe for in situ hybridization was transcribed using a DIG RNA Labeling Kit (Roche Diagnostics) following the manufacturer’s protocol. Riboprobes were purified by Micro Bio-Spin Columns P-30 Tris RNase free (Bio-Rad). Paraffin-embedded sections of newborn bone/growth plate were hybridized to digoxigenin-labeled riboprobes (100 ng riboprobe per slide or 50 ng per section). Antigen retrieval was performed by proteinase K incubation (10 μg/ml, 37 °C for 30 min). For detection, tissue sections were incubated with anti-digoxigenin alkaline phosphatase Fab fragments (Roche) for 2 h at room temperature and treated with NBT/BCIP (Sigma) in the dark until a colorimetric change was detected (1 h for Collagen X; overnight for Osteocalcin). Sections were counterstained with 10% eosin and visualized using Keyence BZ-X700 fluorescence microscope (Keyence Corp, Osaka, Japan) at ×10 magnification under bright field.

For preparation of genomic DNA, 2 mL cell culture (OD₆₀₀ 0.5) were sedimented by centrifugation, the cell pellet resuspended in 100 μL salt water and lysed osmotically by the addition of 900 μL TEN-buffer (100 mM NaCl, 1mM EDTA, 20 mM Tris/HCl pH 8.0). Standard phenol/chloroform extraction was performed followed by DNA precipitation using isopropyl alcohol. For Southern analysis 3 μg of genomic DNA cut with AatII were separated on 0.7% agarose gels and blotted on Roti®Nylon membranes (pore size 0.2 μm, Carl Roth GmbH & Co. KG). Southern blots were hybridized in standard hybridization buffer with digoxigenin-labeled DNA-probes and detected by use of Anti-digoxigenin-alkaline phosphatase Fab fragments (# 11093274910, Roche) in combination with the Phototope®-Star Detection Kit (# N7020S, New England Biolabs) according to the manufacturer's protocols. A digoxigenin DNA labeling Kit (# 11277065910, Roche) was used to produce DNA-probes by standard PCR using genomic DNA of Hbt. salinarum R1 as template in combination with the following oligonucleotides: pil-1-probe-fwd and pil-1-probe-rev producing a 1541 bp PCR product; pil-2-probe-fwd and pil-2-probe-rev producing a 686 bp PCR product (see Table S1 for oligonucleotide sequences).