Tissue preparation protocols standardization

There are major differences in tissue processing protocols with variations in the time of immersion in formalin and alcohols. These variables can be controlled and standardized for the individual laboratory but in the case of tissue blocks prepared in other laboratories such variations may significantly affect the staining of tissue antigens. 

RNA is prepared from tissue following a standard protocol for the isolation of RNA from leaves.27 RNA is stored in 70% ethanol without sodium acetate at —70° and dried immediately prior to use. 

Apart from automating the matrix application process, it is critical to evaluate the resulting matrix crystals and coating for analyte extraction, localization, and effect on tissue architecture. Several reports have shown that even for standard analytes mixed with matrices, there is an uneven distribution of the analyte within the MALDI crystals and that sample preparation influences the resulting distribution [18-20], Thus, other active areas of research are focused on optimizing matrix application and sample preparation protocols. 

Quantitative images of elements can be obtained using prepared matrix-matched laboratory standards for calibration as demonstrated in several previous papers (I, 21, 42). The preparation protocol for synthetic matrix-matched laboratory standards for the calibration of imaging LA-ICP-MS for biological tissues is summarized in Fig. 3.3. In general, different synthetic laboratory standards with elements of interest in well-defined concentrations were prepared. For example, five slices of the same biological tissue were spiked with selected standard solutions at... 

There is the potential to establish internal reference standards that will be of use in assessing the effectiveness of sample preparation and fixation, giving an indication of the suitability of the tissue for IHC studies we will address this new area following the discussion of reagent and protocol validation and at the end of this chapter. 

Preliminary experiments were conducted to calibrate the instrumentation used in this study and to determine the amount of particles needed for reliable detection. We used dilutions prepared from purchased silicon dioxide (SiO ) standards (Postnova Analytics) with a known particle size, 70 nm, and a starting concentration of 25 mg/ml of particles suspended in aqueous surfactant. With the available light scattering detector, reliable quantification of the standard could be obtained with as few as 7 x 10 particles per injected sample, which is equivalent to 25 pg of particle mass. Based on these data, our subsequent experimental work used tissue samples containing 1—2 mg of particles. Using particle aliquots greater than 40 times the limit of detection enabled robust quantification in the experiments to develop nanoparticle recovery protocols. 

In the present study, 5- to 6-week-old male C57BL/6 SVJ mice were fed a high-fat diet (45 kcal% fat) or a standard chow diet for 16 weeks, and then were sacrificed by pentobarbital overdose following treatment. Livers were harvested immediately from anesthetized mice and snap frozen at -70°C in liquid nitrogen before analysis. Utilizing the protocol provided for preparation of tissue homogenates (-200 mg liver tissue, 10 pg primary antibody, 50 pL packed beads), we prepared nuclear/mitochondrial and cytoplasmic fractions and enriched for lysine acetylated proteins using an anti-acetyllysine polyclonal antibody. Complexed proteins... 

There are many published TLC protocols that can be used to separate flavonoids that have been extracted from tissue samples and juice. Methods include the use of silica, reverse phase or polyamide plates in a wide variety of solvent systems. Many of these solvent systems are published in a chapter on flavonoids in Kirchner s book on TLC (1967). The flavonoid spots on the developed plates can be visualized in a variety of maners, the most common method being the direct visualization under UV light using plates that have an acid-stable fluorescent indicatior added for visualization at 254 nm. The visualized spots can then be scraped and quantified by UV absorption. Another standard visualization method utilizes a spray of 2% sodium borohydride freshly prepared in methanol followed by fumigation with hydrogen chloride gas (Horowitz 1957). 

The volume of the tissue should not exceed 10% of the volume of TRI REAGENT . Alternatively, rather than using the whole spleen directly, splenocytes can first be prepared from spleen by using standard procedures described for preparation of hybridoma production (see Chapter 1) before total RNA extraction. A spleen from an immunized mouse contains approx. 5 X 10 to 2 X 10 lymphocytes. For total RNA extraction, substitute 10 lymphocytes for a spleen and simply add 10 ml of TRI REAGENT (1 ml of reagent for 10 cells) to the sterile PBS-washed cell pellet. Allow incubation at room temperature for 5 min for lysis, and proceed to Protocol 2. step 2. 

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