Fixed Tissue Immunofluorescence
Fixed Tissue Immunofluorescence - Related Videos
16:31
Simple Elimination of Background Fluorescence in Formalin-Fixed Human Brain Tissue for Immunofluorescence Microscopy
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Cited by 43 •
2017
Background autofluorescence of biological samples often complicates fluorescence-based imaging techniques, especially in aged human postmitotic tissues. This protocol describes how autofluorescence from these samples can be effectively removed using a commercially available light emitting diode light source to photobleach the sample prior to immunostaining.
09:56
Immunofluorescence Microscopy: Immunofluorescence Staining of Paraffin-Embedded Tissue Sections
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2023
Source: Thomas Chaffee1, Thomas S. Griffith2,3,4, and Kathryn L. Schwertfeger1,3,4 1 Department of Lab Medicine and Pathology, University of Minnesota, Minneapolis, MN 55455 2 Department of Urology, University of Minnesota, Minneapolis, MN 55455 3 Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455 4 Center for Immunology, University of Minnesota, Minneapolis, MN 55455 Pathologic analyses of tissue sections can be used to obtain a better understanding of normal tissue...
04:27
Whole-mount Immunofluorescence Imaging: A Fixing and Staining Technique to Evaluate Intact Organoids
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2023
This video describes the technique of immunofluorescent staining of intact prostate cancer organoids and their imaging by whole-mount confocal microscopy. This technique aims to evaluate the ex vivo differentiation capacity of basal and luminal prostate epithelial cells.
10:49
Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens
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Cited by 37 •
2019
A detailed protocol for a six-marker multiplex immunofluorescence panel is optimized and performed, using an automated stainer for more consistent results and a shorter procedure time. This approach can be directly adapted by any laboratory for immuno-oncology studies.
12:21
MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues
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Cited by 11 •
2013
This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.
FAQs
Fixation stabilizes the tissue’s cellular architecture before staining, preserving the structural relationships that give a signal spatial meaning. Permeabilization is then used to make the preserved tissue accessible to antibody probes, allowing them to reach target epitopes. Treating these as distinct stages connects two needs: retaining tissue organization and enabling molecular detection within that organization.
Antibody binding identifies the molecular target, while the attached fluorescent label supplies the readout. When the stained section is exposed to excitation light, the label produces fluorescence at the locations where the antibody has bound. The resulting pattern therefore shows where a target protein or cellular structure is distributed, rather than only indicating that it exists somewhere in the sample.
Multiplex staining allows several targets to be assessed in the same tissue section. This preserves a shared spatial reference, so signals can be compared across the identical tumor region rather than inferred from separate samples. In cancer research, that arrangement can connect tumor markers, immune-cell infiltration, and proliferation with their locations and with one another.
Spatial context shows how molecular features relate to tissue organization. A detected marker can be considered alongside tumor structure, nearby immune-cell infiltration, or patterns of proliferation, rather than treated as an isolated measurement. This relationship-based view helps investigators examine the tumor microenvironment and connect molecular expression with disease progression.
After tissue is fixed and sectioned, the section is permeabilized so antibody probes can access preserved targets. Fluorescently labeled antibodies are then applied for target-specific staining, and the prepared section is examined by microscopy under excitation light. The resulting image is evaluated for the location and distribution of the detected proteins or cellular structures.
Within cancer research, the method can be directed toward tumor markers, immune-cell infiltration, and cell proliferation. It also supports examination of relationships in the tumor microenvironment, where molecular signals and tissue organization can be considered together. These readouts help characterize tumor organization and molecular features while retaining the spatial setting in which they occur.
By mapping molecular expression onto preserved tissue structure, investigators can examine how tumor features are arranged within their tissue context. The resulting spatial information supports analysis of tumor organization, immune-cell infiltration, proliferation, and microenvironment relationships. Connecting these features with their locations helps researchers relate molecular and structural patterns to disease progression.