Incubation protocols

Secondary or link antibody and /or tertiary reagents too concentrated. Repeat staining. Determine correct concentration for each reagent. Incubation temperature and incubation time will affect results. To determine optimal incubation protocol, vary both the time and temperature for each reagent in the IHC staining protocol. 11-13... 

Dellinger M. Apoptosis or necrosis following Photofrin photosensitization influence of the incubation protocol. Photochem Photobiol 1996 64 183-187. 

Fig. 3. Comparison of transfection efficiencies obtained using PolyFect Reagent, a dendrimer-based transfection reagent, and a calcium phosphate-mediated procedure. COS-7 and HeLa cells were transfected in srx-weU plates with a /3-galactosidase expression plasmid using the appropriate protocol. For the calcium phosphate-mediated transfection, 6 pg of plasmid DNA was used and the medium was changed after 5 h incubation. Transfections were performed in triplicate, and transfection efficiency was measured by monitoring the /3-galactosidase activity of extracts obtained from the transfected cells. The amoimt of /3-galactosidase activity in the extracts correlates with the transfection efficiency. Cells were harvested 48 h post-trans-fection...

Classical approaches to plant DNA isolation aim to produce large quantities of highly purified DNA. However, smaller quantities of crudely extracted plant DNA are often acceptable for PCR analysis. Another efficient method for preparation of plant DNA for PCR is a single-step protocol that involves heating a small amount of plant tissue in a simple solution. Several factors influence nucleic acid release from tissue salt, EDTA, pH, incubation time and temperature. These factors must be optimized for different sample substrates. EDTA in the sample solution binds the Mg + cofactor required by the Taq polymerase in the PCR, so the EDTA concentration in the solution, or the Mg + concentration in the PCR, must be carefully optimized. 

After an hour of incubation, the medium is replaced with Neurobasal culture medium. Add 2, 4, and 10 ml of Neurobasal culture medium to the 35, 60, and 100-mm plates, respectively. After 5 to 7 days in culture, the medium must be half-changed, that is, about half of the old medium is replaced with the same volume of fresh Neurobasal culture medium. Halfchange the medium every 5 to 7 days until the neurons are ready to use. For neurons younger than DIV (days in vitro) 7, if necessary, the medium can be completely replaced without affecting cell viability. In contrast to Brewer s protocol (Brewer et ai, 1993), we do not include 25 /iM glutamate during the first four days of culture and find no adverse effect on neurons. 

After 30 to 60 min incubation at 37°, 20- to 40-/d aliquots from each reaction mixture are spotted on 3MM paper discs that are dropped into 10% ice-cold TCA and processed according to the hot TCA procedure (see later). If the translational product is to be quantified only immunologically, the radioactive precursor ( [14C] phenylalanine) is replaced by nonradioactive phenylalanine. Examples of experiments in which the preceding protocol has been used are shown in Fig. 12.3A.B. 

In a study involving decalcified FFPE rat joint tissue sections and a variety of AR methods, Wilson et al.32 reported successful application of 0.2 M boric acid at pH 7.0 as the AR solution combining a low-temperature incubation (60°C for 17 h). The principal advantage of this AR protocol was that it minimized lifting or loss of decalcified hard tissue sections from charged slides. Their basic approach for establishing an optimal AR protocol was a test battery as described above. In a separate series of studies, based upon prior... 

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