After a prehybridization step in Hybridization Cocktail 50% Formamide (AMRESCO, Solon, OH) for 1 h at 58 [degrees]C, colonies and hemocytes were incubated with sense and antisense probes (2 [micro]g/ml biotin-labeled riboprobe
in Hybridization Cocktail) overnight at 58 [degrees]C.
Briefly, 6 [micro]m thick FFPE tissue sections were dewaxed and rehydrated, incubated with proteinase K, postfixed, prehybridized, and hybridized with a 542-nt antisense CTGF DIG-labeled riboprobe
for 16 h at 70[degrees]C.
Polyvalent detection by using the non-radioactive molecular hybridization technique has been performed in two different forms: first, by a mixture of the specific single probes in the hybridization solution, and secondly, by using a unique riboprobe
, called polyprobe, that contains partial nucleic acid sequences of different viruses cloned in tandem (PEIRO et al., 2012).
All activity tests were made with this template, except in Figures 4(b) and 4(c) where the template pMAS30 (plasmid encoding upstream lacZ riboprobe
), digested with SacI, was used.
The cRNA product was reamplified, cleaned (QIAquick PCR Purification Kit; Qiagen, Valencia, CA), and transcribed using the Riboprobe
In Vitro Transcription System (Promega Corp., Madison, WI) according to standard protocol.
To investigate OMT downregulation of the endogenous OMT mRNA steady state levels, northern blot analysis was performed with an omt antisense riboprobe
with the third leaf of those five plants that maintained reduced OMT activity at the 15-leaf stage.
No antigens or RNA were detected by reacting hyperimmune mouse ascitic fluid or riboprobes
with uninfected Vero E6 cells or by reacting ah unrelated hyperimmune mouse ascitic fluid or riboprobe
with SARS-CoV infected cells.
For detection of WNV nucleic acid, an antisense digoxigenin- labeled riboprobe
complementary to nt 4966-5439 of WNV strain NY1999 was generated from plasmid pWNNY-88B-14 (W.I.
The deleted vector was transcribed by the Riboprobe
in vitro Transcription System (Promega).
To determine if the abnormalities in neural crest-derived melanocytes and increased apoptosis in neural regions could be attributed, at least in part, to changes induced in the expression of the neural crest-specific transcription factor Xslug, nonisotopic in situ hybridizations with a digoxigenin-labeled riboprobe
directed against Xslug cDNA (Mayor et al.
In situ hybridization with an usherin-specific riboprobe
(nN2A) confirms that usherins are expressed both in planulae and in juvenile polyps (Fig.
A digoxigenin-labeled antisense riboprobe
was synthesized from the inducible zebrafish hsp70 polymerase chain reaction fragment previously cloned by Lele et al.