05 Apr On Grid Immunogold labeling (IGL)
Immunogold labeling (IGL) or immunolabeling is a powerful methodology for localizing structures in biological specimens.
Immunolabeling tissue sections on TEM grids using “droplet” methods requires the timed transfer of multiple grids through a dozen reagents in a process that can take hours or even a full day. As a result, microscopists experience multiple challenges:
- Difficultly achieving consistent reagent timing, which limits experimental reproducibility
- Grids are easily damaged, lost, or mixed up
- IGL reagents exposed to air can evaporate, precipitate or become contaminated
- IGL reagents and antibodies are expensive
- The mPrep System addresses these challenges:
- Simultaneous processing assures identical reagent timing and reproducible results
- Encapsulated grids are identified and protected from loss or damage
- Enclosed capsules limit air exposure
- mPrep/g capsules require only 20 ul per grid
Automated IGL with the mPrep ASP-1000
With the ASP-1000 the entire immunolabeling process is automated. Simply set up the reagents, start the program, and walk away for the rest of the day or overnight. Use with mPrep/g capsules for grids, and mPrep/s capsules for tissue en bloc IGL.
Manual IGL with a multi-channel pipettor
Using a multichannel pipettor ensures that reagent incubation timing is identical for all grids. Because grids are safely encapsulated reagent exchanges are fast and easy. Use with mPrep/s capsules for tissue en bloc IGL.
6 nm IGL, endosome complex, C. elegans
6 nm IGL, endosome complex, C. elegans
Illustrates automation of immunogold labeling using the mPrep ASP-1000 automated specimen processor. Researchers at the University of Wisconsin prepared C. elegans tissue sections on TEM grids and then used the ASP-1000 to immunolabel an endosomal sorting component with 6 nm gold. The entire 7.5 hour immunolabeling protocol was automatically accomplished. This note also illustrates automated application of 10 nm gold fiducial markers onto thick sections for tomography.
mPrep/g capsules to hold TEM grids
mPrep/f couplers to prevent introducing reagents into the ASP-1000 or pipettors
ASP-1000 for automated immunogold labeling
Pipettor (such as a Gilson P200 volume) to enable reagent pipetting into mPrep/s capsules
mPrep/s capsules to hold tissue samples for en bloc IGL
Microwell plates to hold reagents
Foil seals to cover reagents until use
Example: 1.5 mil thick pierceable AlumaSeal II Film for PCR plates (Contact us for more information)
Fully-automated Immunogold Labeling of Resin Embedded Specimens and On-grid Deposition of Gold Fiducial Particles (Microscopy & Microanalysis 2016)
Preparing LR White Embedded Tissue with mPrep/s Specimen Capsules (Microscopy & Microanalysis 2017)
Optimization of Automated Immuno EM for Both Pre- and Post-Embedding Labeling (Microscopy & Microanalysis 2018)
Neuraminidase 1-mediated desialylation of the mucin 1 ectodomain releases a decoy receptor that protects against Pseudomonas aeruginosa lung infection (J Biological Chemistry 2019)
This 18-minute webinar (broadcast 25 September 2019) presents how the ASP-1000 automates the preparation of biological specimens for TEM and SBEM (serial block face SEM) to provide walk-away convenience, reduced processing time, and easy clean-up. The webinar also discusses how the ASP-1000 provides pre-programmed protocols for preparing multiple tissues and for immunolabeling, and how users can easily modify existing or create new protocols.
Instructions for using mPrep/g capsules for grid handling and processing.
A graphical display illustrating how to load grids into mPrep/g capsules, and how to use pipettors to stain or label grids. Use this guide as a lab reference poster.
Protocol for immuno-labeling grids with mPrep/g
mPrep grid products