Vibratome section

Evers P, Uylings HB. Effects of microwave pretreatment on immunocytochemical staining of vibratome sections and tissue blocks of human cerebral cortex stored in formaldehyde fixative for long periods. /. Neurosci. Methods 1994 55 163-172. 

Preembedding hybridization where permeabilized cells or vibratome sections are hybridized, and then postfixed and embedded as described in Section 3.1.2. 

ANTIGEN RETRIEVAL IN NEURONAL TISSUE SLICES BEFORE VIBRATOME SECTIONING... 

Fig. 13.2. Preferred association of LCM with tumor cells in vivo. Vibratome sections of brain of rats bearing 9L tumor that have been injected i.v. with diO-LCM, and sacrificed 2 min later. Sections in panels A and C were stained with TR-conjugated WGA. Specimens in panels B and C were observed by fluorescence microscopy using fluorescein optics. The scale bar represents 100 pm. (Taken from ref. 531.)...

Fig. 13.3. This figure demonstrates the distribution of fluorescently tagged LCM in brain parenchyma analyzed by confocal laser scanning microscopy. Rats bearing 9L tumors were administered diO-LCM and sacrificed 2 minutes later. Vibratome sections were counterstained with TR-WGA which binds to tumor cells and distinguishes the tumor area from the surrounding normal tissue. Comparison of the area stained with TR-WGA (tumor cells) (B) and that stained with diO (A) indicates that LCM were associated with a large portion of the tumor. (Taken from ref. 531.)...

Fig. 59. 5 -Nucleotidase immunohistochemical staining of rat cerebellum. A. Immunofluorescence. B. PAP-method. Enzyme activity is predominantly found within the molecular layer on Bergmann glial fibers (long arrows). Purkinje cells are surrounded by fine rims of reaction product (small arrows). Within the granular layer 5 -nucleotidase activity is diffusely scattered between granule cells (arrow heads). Vibratome sections. C. Longitudinally sectioned Bergmann glia cell processes (B) of the molecular layer of rat cerebellum. Fine DAB reaction product is located on adjacent membranes of these processes (arrows). Bars in A,B = 50 /tm, in C = 0.5 nm. Schoen et al. (1987).

Ma, B., Jablonska, J., Lindenmaier, W., and Dittmar, K. E. J. (2006) Immunohistochemi-cal study of the reticular and vascular network of mouse lymph node using vibratome sections. Acta Histochem. 109, 15-28. 

Fig. 8.6 Immunohistochemical localization of the neuropeptide SIFamide in the brain of the giant robber crab Birgus latro (Harzsch and Hansson, unpublished data black-white inverted fluorescent images), (a) Horizontal vibratome section of the median brain. The olfactory lobes (OLs) and the hemiellipsoid bodies (HE) dominate the brain, (b) Detail of the hemiellipsoid body showing its layered structure, (c) The OL seems to be composed of three units...

Incubation of Vibratome Sections in Secondary Antibody, Immunoperoxidase Deveeopment, and Initial Screening by Light Microscopy... 

Adjust the vibratome section thickness to 400 pm and the amplitude of vibration to maximum (Vibratome series 1000, Technical Products International). 

Wash the whole tissue (or thick vibratome sections) in cacodylate buffer, 1x10 mm. 

Pretreatments to allow better access of antibodies to antigenic sites are critical when using whole tissues and thick vibratome sections. Some tissues are less penetrable than others as can be revealed empirically. 

A rapid and sensitive alternative to the PAP/ABC protocols presented in Section 3.4. is to use peroxidase-conjugated F(ab) orF(ab )2 fragments of IgGs recognizing the primary antibodies. In our hands, this approach has been especially valuable for wholemounts and thick vibratome sections in which antibody penetration is limited. 

The general strategies in performing immunocytochemistry of amino acids are essentially the same as those for any other molecule. Immunolabelmg may be performed on sections of embedded tissues, on frozen or Vibratome sections, and on wholemounts, with each approach having its own advantages and disadvantages. 

The procedures outlined in this section may be applied without modification when immimolabeling cryostat sections. Notes are cited to explain modifications of the protocols that may be applicable when immunolabeling vibratome sections, wholemounts of retina, wholemounts of invertebrate ganglia, or cultured cells. 

The cleanest signals are generally obtained using antibodies labeled with fluorescine isothiocyanate (FITC) or carbocyanine dyes (Cy 3). If possible, avoid antibodies labeled with rhodamine (TRITC) or, to a lesser extent, Texas red, since in our hands at least, these tend to yield higher levels of background labelling For Vibratome sections, increase the incubation tune to 3-4 h. For retina or other wholemounts, incubation times on the order of 8 h may be necessary. Likewise, for cultured cells, secondary antisera incubation tunes may need to be extended, this will have to be determined empirically. 

Wash times need to be extended for Vibratome sections, wholemounts, and cultured cells. For Vibratome sections, wash in three changes of PBS over 1 h For wholemounts, wash in three changes of PBS over 2-3 h, whereas for cultured cells, give three changes over 30 rmn. 

A lO-mm incubation m the peroxidase substrate is also usually adequate for vibratome sections and for wholemounts. For cells, the length of incubation in the peroxidase substrate can be determined by periodically examining the cells under a microscope. The reaction may be stopped when a suitable signal has been attained... 

Antibody dilution has to be established empirically The final dilution depends on the detection system used. Our rabbit anti-cGMP antisera are diluted 1 300 v/v in TBS-T in combination with a secondary goat or sheep antirabbit antiserum conjugated with fluorescein-isothiocyanate (or CY3) as a reporter molecule. Furthermore, using Vibratome sections antisera can, in general, be diluted 4-10 times farther than using cryostat sections. 

Fig. 4. Electron microscopic immunocytochemical localizi.Uion of GAD in the inner plexiform layer of rat retina. A rat was perfused with 600 niL of 4% paraformaldehyde and 0.2% glutnraldehyde mixture in O.IM phosphate buffer, pH 7.4, containing 8.5% sucrose and 0.01% CaCl2- A 50-pm section obtained from the vibratome sectioning was stained with antiserum against GAD. An unstained bipolar terminal (B ) (at the center) makes synapses to three stained amacrine (Ai) terminals, one unstained possible bipolar terminal (Bj.,), and one unstained gan-gbon cell dendrite (G). A typical bipolar dyad containing the synaptic ribbon (arrow head) and its postsynaptic, stained, amacrine terminal (A ), and unstained ganglion cell dendrite (G) is clearly discernible. Those three stained amacrine terminals (A,) also make direct contact to each other although there are no visible synapses. Other stained (A ) and unstained (A2) amacrine processes are also noted. Reaction product in each stained terminal is seen to be assiKiated mainly with synaptic t esicles and synaptic membrane (counterstained with lead citrate), (x 34,000). Bar = 1 ixm.

Fig. 3. Confocal immunofluorescence showing RP2 localization in photoreceptors of the human retina. An 80-pm Vibratome section of human retina stained with rabbit hRP2-337-350 antiserum was detected with Cy3 conjugated donkey anti-rabbit and visualized by optical sectioning using a Zeiss LSM510 confocal microscope. OPL outer plexiform layer ONL outer nuclear layer OLM outer limiting membrane IS inner segment OS outer segment. Scale bar is 10 pm.

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