Work

A protocol for immunostaining of macrophages in whole-mount mouse cornea, conjunctiva, and lacrimal gland

Year: 2024

Type: article

Abstract: The healthy lacrimal functional unit contains a resident macrophage population. Here, we present a protocol for immunofluorescent staining of macrophage markers, CD11b, F4/80, and CD206, in whole-mount mouse cornea, conjunctiva, and 50-μM-thick lacrimal gland section. We describe steps for dissection, fixation, permeabilization, and blocking. We then detail procedures for the detection and spatial localization of macrophages through immunostaining and confocal imaging. This approach circumvents the need to obtain thin tissue sections and acquire macrophage images from each tissue section. • Steps for surgical isolation of mouse conjunctiva, cornea, and lacrimal gland • Macrophage immunostaining in whole-mount mouse conjunctiva and cornea • Preparation of 50-μm cryosection of lacrimal gland and macrophage immunostaining • The z stack confocal imaging of immunostained tissues Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. The healthy lacrimal functional unit contains a resident macrophage population. Here, we present a protocol for immunofluorescent staining of macrophage markers, CD11b, F4/80, and CD206, in whole-mount mouse cornea, conjunctiva, and 50-μM-thick lacrimal gland section. We describe steps for dissection, fixation, permeabilization, and blocking. We then detail procedures for the detection and spatial localization of macrophages through immunostaining and confocal imaging. This approach circumvents the need to obtain thin tissue sections and acquire macrophage images from each tissue section.

Source: STAR Protocols

DOI: 10.1016/j.xpro.2024.103444

Authors: Saleh Alfuraih, Dhruv Patel, Wonsuk Choi, Karthikeyan Ramasamy, Rais Ansari, Ajay Sharma

Institutions: Northern Border University, Chapman University, Nova Southeastern University

Language: English

Topic: Ocular Surface and Contact Lens

Subfield: Public Health, Environmental and Occupational Health

Field: Medicine

Domain: Health Sciences

SDG: Good health and well-being

Open Access status: gold

Estimated APC paid: $1,500