Introduction

Tissue microarrarys (TMAs) permit the simultaneous comparison of the expression of a target gene across multiple tissues. TMAs have been widely used to identify disease marker genes (1,2,3), analyze cancer pathogenesis (4,5,6,7), screen new drugs, and validate drug targets (8,9). Currently, most TMAs are made by coring cylindrical tissues from paraffin-embedded donor blocks and transferring cores into a paraffin-recipient block (10). Although paraffin-embedded archived tissues have been widely used to prepare TMAs, some researchers are also using fresh frozen clinical tissues to make TMAs (11,12). Fresh frozen tissues may provide better starting materials to make TMAs. Recently, an optimum cutting temperature (OCT)-embedding method (13) was developed to prepare TMAs from fresh tissues. In this approach, fresh tissues were embedded in OCT in dry ice without fixation. The embedded tissues were cored and transferred into an OCT-recipient block for cryostat sectioning. In this protocol, the OCT matrix required all processing to be performed in dry ice. It was reported that dry ice frozen tissues may be too hard and brittle, making them difficult to core properly (14); also it may not be easy to pre-drill array cavities in the dry-ice frozen OCT recipient block (15) using arrayer machine. In this study, we designed a different method to prepare TMAs for frozen tissue samples. The method is centered on the design and use of a new recipient block that contains preformed array cavities. This recipient block can be used to array tissue cores at either room temperature or low temperature (−5° to −10°C). The new recipient block is fundamentally different in design and composition from both paraffinrecipient blocks (10) and OCT blocks (13). After arraying frozen cored samples into the block, the block can be sectioned immediately using a cryostat microtome. TMAs made using this method maintained the integrity of RNAs and proteins. Our method provides an alternative tool to make high-quality TMAs from frozen clinical tissue samples.

Materials and Methods

Design of the Array Recipient Block

A critical component of our method is the preparation of a new recipient block used to array cored frozen tissues. The matrix of the new block contains the following components [all obtained from Sigma-Aldrich (St. Louis, MO, USA)]: 0.2% formalin, 20% gelatin, 10% sucrose, 1% agarose, and 1× phosphate-buffered saline (PBS). The formalin is used to inhibit the growth of fungi and bacteria inside the pre-made gel matrix. Sucrose and gelatin were used so that the recipient block can be sectioned smoothly and easily. Array cavities in the block are made using a metal stamp mold and a small plastic box that acts as a jig to hold the mold in place (Figure 1A). The plastic box is used to retain the molten matrix, and the stamp mold with 8×12 steel pins is used to form the array cavities. Pins are 15 mm in length with a diameter of 1.5 mm. To prepare the recipient block, the mixture was melted by heating in a microwave oven and then poured into the cubic box. The steel mold is inserted into the molten matrix. The matrix is allowed to solidify, and the steel mold is manually removed from the matrix. Removal of the steel mold with 96 long pins then forms 96 long, cylindrical array cavities in the block.

Figure 1. The instrument and prepared tissue microarray (TMA) slides. (A) (Click to enlarge)

Preparation of Tissue Microarray Slides

Different tissues, including fresh frozen mouse and human clinical tissues, have been tested for preparing TMAs. Frozen tissues were trimmed into small cubic blocks, followed by embedding in OCT compound in dry ice. Coring sites were selected by either surface marking of the frozen blocks or overlay of stained sections described previously (10,13). A cylindrical thinwall needle (5.4-cm-long and inner diameter of 1.53 mm) and plunger (5.5-cm-long and inner diameter of 1.53 mm) were used for coring tissues and for transferring cored samples into array cavities in the recipient block, respectively. The coring needle has a flanged inner-tip chamber design so that the cored tissues have the uniform cylindrical column dimensions. Manual coring of tissues was conducted. The diameter of cored tissues was slightly larger than array cavity, which allowed the cored tissue to fit snugly inside the cavity to facilitate tissue sectioning. The coring and transferring was done at −5° to −10°C inside the cryostat microtome. This worked perfectly, and temperatures were well-balanced. At this temperature, it is easy to core and transfer tissue cores without compromising morphology and RNA/protein quality. The cored tissues will not stick on the coring tube. After the recipient block was filled with cores, the block was then transferred into a cryostat microtome for sectioning. TMA sections at 6–10 µm were cut at −20°C and mounted on pre-coated glass slides. Prepared TMA blocks can be also preserved in the freezer at −80°C for months without compromising the quality of RNA, protein, and morphology.