Gestational Regulation of Granulocyte-Colony Stimulating Factor Receptor Expression: MATERIALS AND METHODS(2)

Immunohistochemistry

Immunohistochemical localization of G-CSFR, cytoker-atin 18, or mouse IgG was achieved by the use of monoclonal antibodies (mAbs) LM832 (G-CSFR) or JMB2 (cy-tokeratin 18), or mouse IgG, and the alkaline phosphatase-anti-alkaline phosphatase (APAAP) method as described previously.

RPA

A 496-basepair (bp) BamHI/EaeI fragment of G-CSFR2145-2722 cDNA, which lacks 81 bp between nucleotide positions 2209 and 2290, was obtained from pQB3s (kindly donated by Dr. S. Nagata Osaka Bioscience Institute, Japan). The fragment, corresponding to the membrane-bound receptor lacking a 27-amino acid insertion in the cytoplasmic domain of the mature polypeptide, was cloned into the BamHI/EagI site of pBluescript 2 KS+ (Stratagene, La Jolla, CA) by the use of T4 deoxyribonuclease ligase (MBI, Sunderland, UK), generating the clone pBSG-CSFR. The sequence of the insert was confirmed using Sequenase 2 (Amersham Life Science Ltd., Little Chalfont, UK). antibiotic levaquin

The G-CSFR (595 bases) and human glyceraldehyde-3-dehydrogenase (GAPDH, 226 bases) cRNA probes for use in RPA were generated by the use of T3 polymerase and the Promega Transcription Kit (Promega Corporation, Madison, WI) according to manufacturer’s instructions and were purified by centrifugation through Qiaquick PCR purification columns (Qiagen, Valencia, CA).

This entry was posted in Human Placenta and tagged Human Placenta, Receptor Expression, Stimulating Factor.