Fig 1.
Common examples of section damage (downloaded from the Allen Reference Atlas).
Obtaining undamaged cryostat sectioned material is difficult, especially for relatively thin sections (20–25μm). A scan through the Allen Reference Atlas, one of the most commonly used anatomical references of the mouse, turns up examples of section damage (arrows). The three most common sources of damage are: folded areas (solid black arrows), as in A, B and C; torn sections (arrowheads, as in C), and sections with missing areas (dotted arrows, in D). Allen Reference Atlas, coronal Level 73 is shown in (A); level 83 is shown in (B); level 84 is shown in (C); and level 96 is shown in (D). Scale bars are 1mm.
Table 1.
Customized Parameters of Tape-Transfer Curing Platform.
Fig 2.
An exploded-view of the redesigned tape-transfer system curing platform.
The platform is composed of: the base enclosure, the printed circuit board (PCB) with soldered UV-LEDs, a glass plate, a top cover, foam spacers, a foam pad, and a lever door. With the exception of the PCB, the glass plate, the foam spacers and pad, all other components were 3-D printed in VeroWhite, through Vistatek Corporation (Vadnais Heights, MN). The base enclosure has side access, to allow for connecting the ground and power terminals of the UV-LEDs. The enclosure as illustrated at bottom left allows for sections to be collected on two slides and for the slides to be cured simultaneously. A front view of the platform is shown on the top left. When lowered, the lever door and foam pad are intended to shield the operator from the UV activation and to apply a mild pressure on the slides, which facilitates the curing process.
Fig 3.
Production use of the tape-transfer system to section perfused mouse brains.
This figure shows the use of this system to collect coronal sections of a perfused mouse brain. Step1, remove a frozen brain block from -80°C storage. Allow the block to acclimate in the cryostat for 30 minutes and then freeze the block onto the specimen chuck. The surface to be sectioned, should be facing up (towards the operator). Step2, attach the chuck and block to the specimen holder and orient relative to the knife. Step 3 and 4, trim the block at 50 or 100μm until the first indication of tissue. Step 5, peel the protective film (white arrow) from one chilled tape-window. Step 6, lightly attach the exposed adhesive surface of the tape-window to the blockface. The black line in the lower part of the tape-window should align with the bottom edge of the block. Use the hard-roller to further press and adhere the tape onto the block. Step 7, while supporting the bottom part of the tape-window, cut a 20μm section from the block. Step 8 and 9, move the tape-window and attached section, to the back of the cryostat and then onto a coated glass slide on the curing platform. Use the soft-roller to smooth the tape. Step 10, repeat Steps 5–9 until a total of 3 tape-windows are attached to each slide. Step 11, close the lever-door and apply the UV for 8–10 seconds. Reflections of the UV can be seen in the metal surface of the cryostat and through the open vents. Step 12, peel the tape-windows from each slide at a slight angle and remove the slides from the cryostat.
Fig 4.
Thumbnail images of serial sections, cut from a perfused mouse brain in three standard sectioning planes.
Selected rows of serial sections cut in the coronal (top), sagittal (middle), and horizontal (bottom) planes. Sections were collected using the tape-transfer method, processed for Nissl stain, coverslipped, and imaged. In all cases, the sections appear of consistently high-quality. A detailed examination of all sections from these three series is shown in Table 2.
Fig 5.
Tape-transfer cut sections are directly congruent to the blockface.
A sagittal Nissl-stained section (approx. Lateral 1.725 mm) collected by the tape-transfer method. Arrows indicate separated pieces of brain (cerebellum and olfactory bulb) which nevertheless remain in an intact anatomical relationship, as in the original whole brain. Compare orientation with the inset of the frozen blockface at lower right (small arrows mirror large arrows in the histological section). Scale bars are 5mm.
Table 2.
Rate of various tissue defects compared between Tape-Transfer sectioned material and published images from the Allen Reference Atlas.
Fig 6.
Compatibility of tape-transfer and the detection of injection GFP/RFP signal, compared to conventional sectioning methods.
Coronal sections of a perfused mouse brain, that was injected with AAV-GFP and AAV-RFP and cut using tape-transfer (A-C); direct on-slide (D-F) and free-floating (G-I) technique. Low-magnification view of each section is shown in A, D and G. Sections correspond to ARA coronal levels 66 to 67.Higher-magnification view of the labeled cells and fibers in the left-hemisphere is shown in B, E, H and in the right-hemisphere in C, F, I. The sections are shown as they appear on the slide, without any adjustment. No major difference in fluorescence intensity is evident, for either the AAV-GFP or AAV-RFP. Note, the tape-transfer cut section (A-C) appears in a different left-right orientation than the other two sectioning modes. This is because the tape-transfer method preserves the orientation of the section as it appears on the blockface (Fig 5). The notation LH and RH is used to indicate the left or right hemisphere, as it relates to the brain. Scale bars are: 1mm (A, D, G) and 100μm (B, C, E, F, H, I).
Fig 7.
Histochemical detection of BDA sections, cut using the tape-transfer system.
A coronal section (approx. Bregma -1.055 mm) of a perfused mouse brain, with a cortical injection of BDA (asterisk). The contralateral projections can be seen in clear detail at higher-magnification in B. The staining is of high-quality and labeled fibers are obvious in the corpus callosum (CC) and in the projection site. Scale bars are: 1mm (A) and 100μm (B).
Fig 8.
Histochemical, Immunohistochemical staining and the use of tape-transfer to section rat and macaque tissue.
Low-magnification views of coronal sections, from mouse brains, reacted for (A) myelin Gallays silver stain technique), (C) tyrosine hydroxylase (TH), and (D) wisteria floribunda lectin (WFA). For each image, the arrow points to the region shown at higher magnification in (B), (E), and (F). Tissue with native fluorescent expression is shown in (G) (and, higher magnification, H) for a mouse brain expressing Cre-dependent GFP in GAD2 cells. Coronal sections of rat and monkey brain (frontal cortex of one hemisphere, medial surface at the left), collected by the tape-transfer method are shown in (I) and (J). All sections are 20μm thick. Scale bars are: 1mm (A, C, D, G, I); 200μm (B, E, F); 50μm (H); 6mm (J). The stereotaxic coordinates of the low-magnification sections are relative to Bregma (Allen Reference Atlas): +.145mm (A); -2.055mm (C); -1.555 (D); +.545mm (G); -2.85 ([12] (I)).