We can generate paraffin-embedded tissue arrays containing from 60-300 tissue spots. These can be custom-made according to the needs of the investigator, and are suitable for immunohistochemistry and in-situ hybridization.
Immunohistochemistry for rhodopsin, on WT and retinal degenerate (rd-1) retina, from postnatal day 1 through 180.
Tissue arrays - a brief overview
Tissue arrays provide the means to assemble multiple samples for simultaneous evaluation of protein or RNA expression. Tissue samples are selected according to the experimental design, for example by organ, age, genotype, mouse strain, or experimental condition. Tissue is fixed and embedded to generate a paraffin donor block, from which an HE stained section is cut. The section is used to identify the region of interest. A “punch biopsy” of the site is taken from the donor block, and placed in the grid of the recipient block. Once the grid is filled, sections can be cut to generate tissue arrays similar to the one illustrated above. Depending on the size of the punches used (0.6mm, 1mm, 1.5mm or 2mm), from 60 –300 tissue samples can be arrayed on one slide. As the tissue samples become exhausted at different rates when sections are cut from the array, several tissue samples per data point are recommended. For example, the array illustrated above has five tissue spots per time point per genotype. In addition, these have been collected from 2-3 different animals to overcome the pitfall of skewed data arising from individual variation.
Several steps need to be taken prior to production of the array:
The purpose of the array will determine which tissue samples will be collected. For example, if the array is required to identify organ specific expression of a protein, only one mouse of a selected strain, gender or age may be required. Alternately, if the experiment evaluates gene expression through development, or through the course of the disease, tissues from many animals may be required. Please feel free to contact us to discuss the design of the array.
Preparation of donor tissue
Tissues may be fixed in 10% Formalin, Feketes Acid Alcohol or Bouin’s solution. Please refer to Fixation Protocols to select the most appropriate method. Bony tissues will need to be decalcified (see Fixation Protocols) unless they are fixed in Bouin’s solution. Exposure of tissues to acid may reduce signal when in-situ hybridization is performed. Once a full set of samples has been collected, bring these to LSOG 118 for cutting-in, embedding and processing.
Generation of donor blocks, and selection of donor site
Tissues will be embedded in paraffin and an HE section cut for each block. The slides are returned to the investigator, who then marks the region of interest on the slide (a dot with a black marker will suffice). This is matched up with the block before it is punched.
Generation of the array
The array design is constructed in table format by the investigator. This is usually made in a program such as Excel or Word, and lists the coordinates of each spot in grid form. The identity of the donor block is given for each coordinate – in this way a reference key to identify each spot on the array is established. The array block is then constructed, and sections cut. Depending on the thickness of the tissue, 20-40 good sections per block can be obtained before data points begin to be lost.
Please fill out a Tissue Array Request Form, and we will contact you to arrange a time to discuss the array.