Improved Sectioning and Berlin Blue Staining of Whole Human Brain

Brain sectioning has been improved through gelatin embedding so that more than forty precisely oriented serial sections can be obtained from a single brain. Since the embedding gelatin requires no fixation, it can be removed from the sections prior to staining by simple warming. The reduction of Berlin blue dye commonly observed after staining by the Mulligan method has been found to be at least partly due to light in the UV to near UV range. Dye reduction is significantly inhibited by postfixation in 25% acetic acid.     

Wool Fibres: Sectioning and Staining, Differentiation of Ortho and Paracortex

A double embedding technique for tangential sectioning of hair and wool fibres is as follows: The cleaned fibre bundle is attached to a U-shaped, 16 gauge, tinned-copper wire frame with collodion adhesive, soaked in 6% nitrocellulose for 1 hr, and treated with chloroform for 2 hr. The hardened bundle is then cut fom the wire support and embedded in paraffin-beeswax, 95:5. Sectioning is at 6-8 μ. The use of 2% orange G or saturated aqueous picric acid for quantitative study of the fibres, and the demonstration of wool fibre cortical fractions by staining with polychrome methylene blue after oxidation of the sectioned fibres in a solution of formic acid (98/100 w/v) 25 ml; distilled water, 65 ml; and H₂O₂ (30% w/v), 10 ml, for 1 hr, is recommended.     

Epoxy Embedding, Sectioning and Staining of Plant Material for Light Microscopy

By using a formula which gives a relatively soft epoxy embedding medium, it is possible to cut sections of plant material with a sliding microtome equipped with a regular steel knife. Blocks having a cutting face of 10 × 10 mm, giving sections of 4-10 μm, can be used. Tissues are fixed in Karnovsky's fluid, postfixed in 1 or 2% OsO₄, embedded in Spurr's soft epoxy resin, Araldite, or Epon mixtures. 5% KMnO₄, followed by 5% oxalic acid, then neutralized in 1% LiCO₃, are used to mordant the sections. Some of the stains used are Mallory's phosphotungstic acid-hemotoxylin, acid fuchsin and toluidine blue, or toluidine blue. Mounting is done with whichever soft epoxy resin was used in casting the blocks.     

Capsules for Frozen Sectioning of Small Specimens

It was necessary to make sections of small unfixed specimens which had been frozen while still immersed in their normal culture medium. The principal difficulty stemmed from the poor sectioning quality of the frozen culture medium. A capsule is described which has a narrow well in which the tissue specimen fits snugly within a small amount of culture medium. After freezing, the whole capsule is sectioned and the resulting sections, being nearly devoid of culture medium, are of good quality.     

Block Staining with Hematoxylin, Gelatin Embedding and Serial Sectioning of Decalcified Bone

Decalcified pieces of bone can be stained prior to sectioning by washing and placing in undiluted Harris' hematoxylin (potassium alum). This stains the borders and contents of Haversian canals, lacunar margins, osteocyte nuclei, cementing lines and canaliculi. Embedding in 15% gelatin gives a flexibility which allows mounting of large numbers of serial sections with a minimum of tearing and folding. Frozen sections from the microtome knife are placed in water-filled depressions of chemists' porcelain spot plates, or other containers in the case of large sections. Long pieces of bone can be cut into smaller blocks, which are placed in additional gelatin. This allows complete sectioning of the block and examination of structures continuing throughout the bone.     

Decalcification of Snail Shells to Facilitate Sectioning of the Whole Organism

This note describes the application of several decalcification procedures used on snail shells to permit histologic sectioning of the intact soft tissues. The configuration of the body parts, the integrity of the cells and their capacity to react to standard staining procedures is not impaired (figures 1, 2). The following bone decalcifers were used in a comparison test: Pereny's method (Luna 1968), nitric acid method I & II (Luna 1968), formic acid-sodium citrate method (Luna 1968), and two commercial products: RDO—DuPage Kinetic Laboratories, Downers Grove, IL 605 15; and Decal—Omega Chemical, Cold Spring On Hudson, NY 10516     

A Rapid Method For Concentrating Small Organisms For Sectioning

Lipp, Walther. Histochemische Methoden. About 24 pages per issue; 3 issues (No. 9, 10 and 11) during 1956. 16 £ 23 cm. R. Oldenbourg, München 1, Germany. DM 30.—for 6 issues; DM 6.—per single copy. (In German)

Molenaar, I. Ontkalking van harde weefsals. Een histochemische studie. Thesis, Rijksuniversiteit te Utrecht, 1957. pp. 124.     

Visualizing Peripheral Nerve Regeneration by Whole Mount Staining

Peripheral nerve trauma triggers a well characterised sequence of events both proximal and distal to the site of injury. Axons distal to the injury degenerate, Schwann cells convert to a repair supportive phenotype and macrophages enter the nerve to clear myelin and axonal debris. Following these events, axons must regrow through the distal part of the nerve, re-innervate and finally are re-myelinated by Schwann cells. For nerve crush injuries (axonotmesis), in which the integrity of the nerve is maintained, repair may be relatively effective whereas for nerve transection (neurotmesis) repair will likely be very poor as few axons may be able to cross between the two parts of the severed nerve, across the newly generated nerve bridge, to enter the distal stump and regenerate. Analysing axon growth and the cell-cell interactions that occur following both nerve crush and cut injuries has largely been carried out by staining sections of nerve tissue, but this has the obvious disadvantage that it is not possible to follow the paths of regenerating axons in three dimensions within the nerve trunk or nerve bridge. To try and solve this problem, we describe the development and use of a novel whole mount staining protocol that allows the analysis of axonal regeneration, Schwann cell-axon interaction and re-vascularisation of the repairing nerve following nerve cut and crush injuries.     

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell ¹.Open in a separate windowClick here to view.^{(64M, flv)}     

Staining OF Embryonic and Small Mammalian Skeletal Systems

The methods commonly used for staining developing bone (Lundvall, 1905; Spalteholz, 1914; Batson, 1921; Dawson, 1926; Richmond and Bennett, 1938; Cumley, Crow, and Griffin, 1939; Williams, 1941), have involved the use of 1% or 2% Aqueous KOH without employing any bleaching agent. The introduction of hydrogen peroxide into the technic proves to be a distinct advantage, and in the concentration here proposed, does not result in the production of fine bubbles. The maceration process can be hastened through the use of an increased concentration of potash in the absence of a fixative, although this in itself is one of the hazards of the technic. However, if the preparations are carefully watched, this difficulty may be largely avoided. The method works admirably and gives a very clear muscle tissue so that the skeletal elements are very sharply outlined. The proposed method (arranged in the form adopted in Staining Procedures) is herewith described.     

A Rapid Method for Sectioning Whole Mature Coniferous Seeds for Histological and Histochemical Study

A method is given for rapidly sectioning whole mature conifer seeds. The procedure consists of soaking seeds in water and allowing the seed contents to swell, whereupon the seeds are sectioned in a cryostat. Sections thus prepared are suitable for histological and histochemical staining.