Use of LR White Resin for Post-Embedding Immunolabelling of Brain Tissue

Gocht, Andreas

doi:10.1159/000147385

Cited by 6 publications

(4 citation statements)

References 13 publications

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“…With an additional few days required to examine multiple EM sections and find the region of interest, this is comparable to standard CLEM methods that involve culturing cells on gridded coverslips as the imaging and processing times are very similar [ 26 ]. The iEM approach using cryostat sections can take up to a day or two longer than for cryo-ultramicrotomy due to the sample processing time but the timeframe is similar to room temperature ultramicrotomy of LR white/Lowicryl type embedded specimens [ 27 , 28 ].…”

Section: Discussionmentioning

confidence: 99%

Correlative light and immuno-electron microscopy of retinal tissue cryostat sections

et al. 2018

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Correlative light-electron microscopy (CLEM) is a powerful technique allowing localisation of specific macromolecules within fluorescence microscopy (FM) images to be mapped onto corresponding high-resolution electron microscopy (EM) images. Existing methods are applicable to limited sample types and are technically challenging. Here we describe novel methods to perform CLEM and immuno-electron microscopy (iEM) on cryostat sections utilising the popular FM embedding solution, optimal cutting temperature (OCT) compound. Utilising these approaches, we have (i) identified the same phagosomes by FM and EM in the retinal pigment epithelium (RPE) of retinal tissue (ii) shown the correct localisation of rhodopsin on photoreceptor outer segment disc like-structures in iPSC derived optic cups and (iii) identified a novel interaction between peroxisomes and melanosomes as well as phagosomes in the RPE. These data show that cryostat sections allow easy characterisation of target macromolecule localisation within tissue samples, thus providing a substantial improvement over many conventional methods that are limited to cultured cells. As OCT embedding is routinely used for FM this provides an easily accessible and robust method for further analysis of existing samples by high resolution EM.

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Section: Discussionmentioning

confidence: 99%

Correlative light and immuno-electron microscopy of retinal tissue cryostat sections

et al. 2018

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“…Ultrathin sections were collected on nickel grids and processed for the immunohistochemical analysis (Isola et al 2017). Though the acrylic resin/UV light method is usually regarded as more sensitive (Gocht, 1992) than the currently used, well established immunohistochemical method, the outcome of the comparisons between isoprenaline-treated and control glands is likely to be the same regardless of method.…”

Section: Transmission Electron Microscopy and Immunogold Techniquementioning

confidence: 99%

Melatonin release by exocytosis in the rat parotid gland

et al. 2018

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Several beneficial effects on oral health are ascribed to melatonin. Due to its lipophilic nature, non-proteinbound circulating melatonin is usually thought to enter the saliva by passive diffusion through salivary acinar gland cells. Recently, however, using transmission electron microscopy (TEM), melatonin was found in acinar secretory granules of human salivary glands. To test the hypothesis that granular located melatonin is actively discharged into the saliva by exocytosis, i.e. contrary to the general belief, the b-adrenergic receptor agonist isoprenaline, which causes the degranulation of acinar parotid serous cells, was administered to anaesthetised rats. Sixty minutes after an intravenous bolus injection of isoprenaline (5 mg kg À1 ), the right parotid gland was removed; pre-administration, the left control gland had been removed. Samples were processed to demonstrate melatonin reactivity using the immunogold staining method. Morphometric assessment was made using TEM. Gold particles labelling melatonin appeared to be preferentially associated with secretory granules, occurring in their matrix and at membrane level but, notably, it was also associated with vesicles, mitochondria and nuclei. Twenty-six per cent of the total granular population (per 100 lm 2 per cell area) displayed melatonin labelling in the matrix; three-quarters of this fraction disappeared (P < 0.01) in response to isoprenaline, and melatonin reactivity appeared in dilated lumina. Thus, evidence is provided of an alternative route for melatonin to reach the gland lumen and the oral cavity by active release through exocytosis, a process which is under the influence of parasympathetic and sympathetic nervous activity and is the final event along the so-called regulated secretory pathway. During its stay in granules, anti-oxidant melatonin may protect their protein/peptide constituents from damage.

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“…Tissue was thoroughly washed in tap water, dehydrated, and embedded in paraffin. Sections were cut at 5-7 µm and processed for immunohistochemistry by using the avidin-biotin complex (ABC) method (Hsu et al, 1981) as described earlier (Gocht, 1992). Briefly, sections were deparaffinized, and thereafter lipid was removed with chloroform for 24 hours (Perentes and Rubinstein, 1985).…”

Section: Optic Nerve Sectionsmentioning

confidence: 99%

Fate of developing astrocytes in the optic nerve of the myelin-deficient rat

Struckhoff

Przyrembel²,

Bähr³

et al. 1997

J. Comp. Neurol.

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There is considerable debate on the development of a glial cell line in the rat optic nerve, which is characterized by the specific expression of the A2B5 and HNK-1 epitopes. This cell line has been assumed to give rise to oligodendrocytes and so-called type 2 astrocytes. However, it is doubtful that the latter cell type really exists in vivo. In the present study, we have addressed this question by investigating the development of astrocytes in the myelin-deficient (md) rat, which is characterized by dysmyelination and loss of oligodendrocytes. Defective oligodendrocytes were observed by the third postnatal day, well before the generation of type 2 astrocytes. Consequently, the number of type 2 astrocytes was reduced in cultures prepared from optic nerves of md rats vs. controls. This finding was not paralleled in vivo; i.e., no dying astrocytes were observed in md sections by conventional electron microscopy. However, immunoreactivity against the HNK-1 epitope was enhanced in md compared to control sections. Ultrastructurally, HNK-1 immunoreactivity was detected predominantly on the axonal surface at astroaxonal contact sites, which were found only at the nodes of Ranvier within controls but extended to the whole axonal surface in md animals. Only a minor portion of the immunoreactivity derived from glial cells, presumably from oligodendrocytes at the paranodal region in controls. Thus, the HNK-1 epitope is not a useful antigen for distinguishing astrocytes in the rat optic nerve. Accordingly, our results do not provide evidence for the existence of specialized type 2 astrocytes in vivo. In vitro, these cells are probably only oligodendrocytes that mimic some astroglial features if grown in serum-containing media.

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Use of LR White Resin for Post-Embedding Immunolabelling of Brain Tissue

Cited by 6 publications

References 13 publications

Correlative light and immuno-electron microscopy of retinal tissue cryostat sections

Correlative light and immuno-electron microscopy of retinal tissue cryostat sections

Melatonin release by exocytosis in the rat parotid gland

Fate of developing astrocytes in the optic nerve of the myelin-deficient rat

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