{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T18:29:42Z","timestamp":1787077782022,"version":"build-2736575974"},"posted":{"date-parts":[[2026,4,6]]},"group-title":"Developmental Biology","reference-count":22,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,4,6]],"date-time":"2026-04-06T00:00:00Z","timestamp":1775433600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"name":"National Institute of General Medical Sciences of the National Institutes of Health","award":["P20GM103499"],"award-info":[{"award-number":["P20GM103499"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,8,14]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>Immunohistochemistry (IHC) is widely used to assess protein expression in corneal tissue, yet staining outcomes are strongly influenced by tissue preparation methods and regional differences within the cornea. This study aimed to systematically compare three processing techniques including paraffin (wax) embedding, wax embedding with antigen retrieval (wax AR), and cryosectioning for IHC analysis in embryonic day 18 chicken corneal tissue. Markers representing key biological functions were evaluated, including progenitor activity (PAX6, P40), tissue architecture (actin), and immune surveillance (TAP1, CD68), across central and limbal regions. Cryosectioning consistently produced the most specific staining for nuclear and antigen-sensitive markers. PAX6 and P40 exhibited strong, nuclear-localized expression in the corneal epithelium only under cryo conditions, whereas wax-based methods resulted in reduced specificity and irregular signal distribution. TAP1-positive immune cells were detectable in the limbal stroma exclusively in cryosections, highlighting improved antigen preservation. In contrast, actin staining, was best preserved with wax AR, and provided superior structural clarity and expected expression patterns across corneal layers. CD68 showed minimal or inconsistent staining in corneal tissue across all methods despite positive control validation. These findings demonstrate that optimal IHC outcomes in corneal tissue are marker-dependent and influenced by preparation methods and regional tissue context. Cryosectioning is recommended for detecting nuclear and immune-related antigens, while wax AR is preferable for preserving tissue architecture. This study provides a practical framework for improving reproducibility and interpretation of corneal immunostaining in avian models.<\/jats:p>\n                <jats:sec>\n                  <jats:title>Highlights<\/jats:title>\n                  <jats:list list-type=\"bullet\">\n                    <jats:list-item>\n                      <jats:p>Cryosectioning preserves nuclear and immunity antigens in cornea<\/jats:p>\n                    <\/jats:list-item>\n                    <jats:list-item>\n                      <jats:p>Wax AR improves structural markers such as actin in corneal tissue<\/jats:p>\n                    <\/jats:list-item>\n                    <jats:list-item>\n                      <jats:p>Staining outcomes depend on marker and tissue preparation method<\/jats:p>\n                    <\/jats:list-item>\n                    <jats:list-item>\n                      <jats:p>Cryosectioning enables detection of limbal immune-associated cells (TAP1)<\/jats:p>\n                    <\/jats:list-item>\n                    <jats:list-item>\n                      <jats:p>CD68 shows limited corneal expression despite positive control staining<\/jats:p>\n                    <\/jats:list-item>\n                  <\/jats:list>\n                <\/jats:sec>","DOI":"10.64898\/2026.03.30.715369","type":"posted-content","created":{"date-parts":[[2026,4,6]],"date-time":"2026-04-06T11:55:16Z","timestamp":1775476516000},"source":"Crossref","is-referenced-by-count":0,"title":["Comparison of immunohistochemistry methods in embryonic chicken corneal tissue"],"prefix":"10.64898","author":[{"given":"Joshua T.","family":"Harkins","sequence":"first","affiliation":[{"name":"Department of Biology, Winthrop University, Rock Hill, South Carolina, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mitchell M.","family":"Hill","sequence":"additional","affiliation":[{"name":"Department of Biology, Winthrop University, Rock Hill, South Carolina, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jena L.","family":"Chojnowski","sequence":"additional","affiliation":[{"name":"Department of Biology, Winthrop University, Rock Hill, South Carolina, 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