Hepatitis B Virus (HBV)

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Quantitative Gonadotropin-Releasing Hormone: MATERIAL AND METHODS(8)

The reaction mix was covered with 50 ^l light white mineral oil, put in the DNA Thermal Cycler 480, and heated to 99°C for 9 min to denature all proteins and to activate the polymerase gold. After completion of 30 cycles of 94°C for 45 sec, 56°C for 45 sec, and 72°C for 60 sec, […]

Quantitative Gonadotropin-Releasing Hormone: MATERIAL AND METHODS(7)

After sequence confirmation, 1 ng of target cDNA was amplified using a modification of a method previously described to construct a competitive cDNA fragment: a floating primer with a sequence complementary to the cDNA between the 3′ and 5′ primer binding sites was designed by attaching this sequence to the reversed complementary sequence of the […]

Quantitative Gonadotropin-Releasing Hormone: MATERIAL AND METHODS(6)

RT For each mRNA to be detected, 19 ^l RT-mastermix was prepared (5 mM MgCl2 solution, single-strength PCR-buffer II, 2.5 |xl DEPC-treated dH2O, 1 mM dATP, 1 mM dCTP, 1 mM dGTP, 1 mM dTTP, 2.5 |xM oligo(dT)16, 20 IU RNase inhibitor [all Perkin-Elmer, Foster City, CA], 100 IU murine leukemia virus reverse transcriptase [Gibco […]

Quantitative Gonadotropin-Releasing Hormone: MATERIAL AND METHODS(5)

RNA Extraction The extraction of RNA from the tissue sample was carried out as described previously with the RNA-STAT-60 reagent (Tel-Test ‘‘B’’ Inc., Friendswood, TX). Briefly, tissue samples were washed three times in PBS (Gibco BRL, Grand Island, NY) to remove blood contamination. buy asthma inhalers One hundred milligrams of tissue was homogenized in 1 […]

Quantitative Gonadotropin-Releasing Hormone: MATERIAL AND METHODS(4)

After tissue digestion, the stromal and epithelial cells were isolated as follows: the cell solution was allowed to settle for 5-10 min, and then the supernatant (stromal-rich fraction) was filtered to a new tube with cell strainer (70-^m nylon; Falcon Plastics, Los Angeles, CA). The settled pellet (epithelial-rich fraction) was rinsed with DMEM. The process […]

Quantitative Gonadotropin-Releasing Hormone: MATERIAL AND METHODS(3)

Immunohistochemical controls were incubated with PBS containing 2% goat serum without primary antibody. To amplify the signal, sections were washed with PBS-T, and then the avidin-biotin alkaline phosphatase-staining method (Vector Lab., Inc., Burlingame, CA) was used. Endogenous alkaline phosphatase activity was inhibited by the addition of levamisole to the buffer used to prepare the substrate […]

Quantitative Gonadotropin-Releasing Hormone: MATERIAL AND METHODS(2)

Tissue Processing Fixed tissue was embedded in paraffin, sectioned, and mounted. Twelve serial sections (6 ^m) from each sample were then prepared for immunohistochemistry, and the first and last sections were stained with hematoxylin-eosin and examined with a Nikon microphot-FXA microscope (Nikon Instruments, Garden City, NY).

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