Isolation of Intact Eyeball to Obtain Integral Ocular Surface Tissue for Histological Examination and Immunohistochemistry

Guo, Dianlei; Liu, Chunqiao

doi:10.3791/60086

Cited by 3 publications

(9 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…2K). This observation thus explains the mutant precocious eyelid opening previously found by Guo (Guo et al, 2019), and led us to further investigate mutant ocular fluid production in time series.…”

Section: Resultsmentioning

confidence: 55%

“…Tear duct dysplasia in a severe Prickle 1 hypomorphic mutant led to epiphora A severe hypomorphic Prickle 1 mutant that is compound heterozygous for the null and hypomorphic alleles was generated (see Materials and Methods, Supplemental Fig. 1A) (Guo et al, 2019;Guo et al, 2018). We estimated that Prickle 1 was expressed less than 25% of WT levels from western blots using limb and brain tissues (Supplemental Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Animal husbandry and experimentation were conducted in strict adherence to the Standards in Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines, with approval from Animal Care and Use Committee (ACUC), Zhongshan Ophthalmic Center, Sun Yat-sen University. The severe Prickle 1 hypomorph mutant mouse line used in this study was generated by crossbreeding a Prickle 1 gene-trap mutant allele (Prickle 1 a/+ ) to a straight knockout allele (Prickle 1 b/+ ) (Guo et al, 2019;Liu et al, 2014;Liu et al, 2013). Mouse genotyping was conducted as described previously (Liu et al, 2014;Liu et al, 2013).…”

Section: Mice and Genotypingmentioning

confidence: 99%

“…For paraffin eyeball sections, the eyeballs including the intact eyelids were dissected and fixed in 4% paraformaldehyde for 24h at 4℃ (Guo and Liu, 2019). The samples were washed 3 times in PBS (phosphate buffered saline), dehydrated through a series of alcohols and 3 times of xylene, then embedded in paraffin and sectioned using microtome (Leica RM 223, Wetzlar, Hesse-Darmstadt, Germany).…”

Section: Histologymentioning

confidence: 99%

“…The Wnt/PCP pathway has been shown to be involved in ocular development and diseases recently. Particularly, a mutation in Prickle 1 in mice causes ocular surface inflammation and precocious eyelid opening (Guo et al, 2019), the primary cause of which has yet to be identified. Additionally, a delayed embryonic eyelid closure was also observed in Prickle 1 mutant mice (Guo et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

Obstructed tear duct causes epiphora and precocious eyelid opening due to disruption of Prickle 1-mediated Wnt/PCP signaling

Guo

Fan

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

The tear drainage apparatus evolved in terrestrial animals serving as conduits for tear flow. Obstruction of tear drainage causes a range of ocular surface disorders. Hitherto, genetics of tear duct development and obstruction has been scarcely explored. Here we report that a severe Prickle 1 hypomorph mouse line exhibited epiphora. This phenotype was due to blockage of the tear drainage by the incompletely formed nasolacrimal duct (NLD) and lacrimal canaliculi (CL). Further analysis revealed that the precocious eyelid opening, previously observed in the same type of Prickle 1 mutants, is also caused by tear duct dysplasia. A comparison of wild type, the Prickle 1 hypomorph and null mutants revealed a dose-dependent requirement of Prickle 1 for tear duct outgrowth. As a key component of a set of six Wnt/PCP core proteins, Prickle 1 usually works together with other PCP components. An investigation of expression of Wnt/PCP core genes demonstrated three of the six PCP components in tear duct, supporting the notion of contextdependent organization of PCP protein complexes. Furthermore, expression of Fgfr2/Fgf10 and p63 genes, mutations of which are associated with NLD and CL hypoplasia in human, were not altered in Prickle 1 mutant mice.Lastly, we showed that Prickle 1 expression in developing tear drainage system is conserved between mouse and human despite anatomical differences. Altogether, the study uncovered how obstruction of the tear drainage could lead to a complex ocular surface disorder, which may have genetic implications in human ocular health.

show abstract

Section: Resultsmentioning

confidence: 55%

Section: Resultsmentioning

confidence: 99%

Section: Mice and Genotypingmentioning

confidence: 99%

Section: Histologymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Obstructed tear duct causes epiphora and precocious eyelid opening due to disruption of Prickle 1-mediated Wnt/PCP signaling

Guo

Fan

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

Malformation of Tear Ducts Underlies the Epiphora and Precocious Eyelid Opening inPrickle 1Mutant Mice: Genetic Implications for Tear Duct Genesis

Guo

Fan

et al. 2020

Invest. Ophthalmol. Vis. Sci.

Self Cite

View full text Add to dashboard Cite

Purpose Obstruction of the tear drainage causes a range of ocular surface disorders. Hitherto, the genetics of tear duct development and obstruction has been scarcely explored, and related animal models are lacking. This study aims to study the potential role of the Wnt/PCP pathway mediated by Prickle 1 in tear duct development and diseases. Methods A severe hypomorphic Prickle 1 mutant was generated. Histology and immunohistochemistry were performed to compare wild type, Prickle 1 hypomorphic, and null mutant tear ducts. In situ hybridization was conducted to identify the signaling components in the developing tear ducts. Three-dimensional (3D) reconstruction was used to detect the human embryonic tear duct. Results Here, we report that a severe Prickle 1 hypomorph mouse line exhibited epiphora. This phenotype was due to the blockage of the tear drainage by incompletely formed nasolacrimal duct (NLD) and lacrimal canaliculi (LC), which also causes precocious eyelid opening. We observed a dose-dependent requirement of Prickle 1 for tear duct outgrowth. An investigation of the expression of Wnt/PCP core genes demonstrated a subset of PCP signaling components expressed in the developing tear duct. Furthermore, Prickle 1 is not required for the expression of Fgfr2/Fgf10 and p63 genes, which are associated with the NLD and LC hypoplasia in humans. Last, we showed that Prickle 1 expression in the developing tear drainage system is conserved between mice and humans. Conclusions The study suggests that malformed tear ducts caused by disruption of Prickle 1 underlies the epiphora and precocious eyelid opening.

show abstract

Tissue block staining and domestic adhesive tape yield qualified integral sections of adult mouse orbits and eyeballs

Ungerer²,

Faßbender

et al. 2021

PLoS ONE

View full text Add to dashboard Cite

The standard histological processing procedure, which produces excellent staining of sections for most tissues, fails to yield satisfactory results in adult mouse orbits or eyeballs. Here, we show that a protocol using tissue block staining and domestic adhesive tapes resulted in qualified integral serial cryo-sections of whole orbits or eyeballs, and the fine structures were well preserved. The histological processing protocol comprises paraformaldehyde fixation, ethylenediaminetetraacetic acid decalcification, tissue block staining with hematoxylin and eosin, embedding, adhesive tape aided sectioning, and water-soluble mounting. This protocol was proved to be the best in comparison with seven other related existing histological traditional or non-traditional processing methods, according to the staining slice quality. We observed a hundred percent success rate in sectioning, collection, and mounting with this method. The reproducibility tested on qualified section success rates and slice quality scores confirmed that the technique is reliable. The feasibility of the method to detect target molecules in orbits was verified by successful trial tests on block immunostaining and adhesive tape-aided sectioning. Application of this protocol in joints, brains, and so on,—the challenging integral sectioning tissues, also generated high-quality histological staining sections.

show abstract

Isolation of Intact Eyeball to Obtain Integral Ocular Surface Tissue for Histological Examination and Immunohistochemistry

Cited by 3 publications

References 0 publications

Obstructed tear duct causes epiphora and precocious eyelid opening due to disruption of Prickle 1-mediated Wnt/PCP signaling

Obstructed tear duct causes epiphora and precocious eyelid opening due to disruption of Prickle 1-mediated Wnt/PCP signaling

Malformation of Tear Ducts Underlies the Epiphora and Precocious Eyelid Opening inPrickle 1Mutant Mice: Genetic Implications for Tear Duct Genesis

Tissue block staining and domestic adhesive tape yield qualified integral sections of adult mouse orbits and eyeballs

Contact Info

Product

Resources

About