The Yokogawa High-Content Spinning Disk Confocal Microscope and CellPathfinder Analysis Software Aid in Antibody Validation

 

Summary

This method, involving mRNA transfection followed by antibody staining and analysis using Yokogawa's CQ1 and CellPathfinder, provides a quantitative approach to validating antibodies. This validation assists researchers in selecting appropriate antibodies for their studies.

Introduction

Antibodies are essential tools in cell-based research, yet reliable methods for validating their efficacy remain limited. This application note describes a straightforward approach using Yokogawa's CQ1 high-content spinning disk confocal microscope, Yokogawa's CellPathfinder analysis software, and Elixirgen Scientific's iPSC-derived astrocytes to validate antibody specificity. This data can assist researchers in determining the efficacy of specific antibodies for future studies. In this experiment, we focus on the validation of antibodies against ALDH1 L1 and GFAP, well-known markers for mature astrocytes. Their respective antibodies must be validated for specific binding to their target antigens to effectively characterize iPSC-derived astrocytes using immunocytochemical staining.

Methods

Cell culture, mRNA transfection, and immunocytochemistry: iPSCs were seeded on 36 wells of a 96-well plate and allowed to adhere and grow for one day. As a positive control, half of the wells (18 wells) were then transfected with mRNA encoding either Aldehyde dehydrogenase 1 family member L1 (ALDH1L1) or Glial Fibrillary Acidic Protein (GFAP), the target proteins. As a negative control, the remaining 18 wells were not transfected. The next day, all wells and cells were fixed and then stained with three different concentrations of antibodies for either ALDH1 L1 or GFAP or with Immunoglobulin (IgG) as a negative control. The nuclei of all cells were stained with Hoechst 33342.

Image acquisition and analysis

Using a Yokogawa CQ1 high-content spinning disk confocal microscope each well was imaged using a 1Ox objective. To cover the entirety of each well, 4 image fields (a 2x2 square) were taken. Nuclei and ALDH1L1 or GFAP antibody positive cells were segmented using Yokogawa's CellPathfinder analysis software. The total number of cells in the well was determined by the CellPathfinder analysis counting the segmented nuclei. In addition, the CellPathfinder analysis used the ALDH1 L1 or GFAP segmentation to report the number of target antibody positive cells. The segmentation of the target antibody also allowed the CellPathfinder analysis to measure and report the mean intensity of the target antibody.

Results

Images were captured using a Yokogawa CQ1 high-content spinning disk confocal microscope. Staining of ALDH1L1 or GFAP was clearly seen in undifferentiated iPSCs transfected with target mRNA (Figure 1A and 1B). The transfection efficiency of ALDH1L1 or GFAP was not 100%, as indicated by the mosaic pattern of the staining. No signal was observed in similarly stained untransfected cells, thus indicating a lack of non-specific binding of the antibodies. Transfected cells displayed no signal when treated with IgG, demonstrating that the antibodies used are specific, and therefore a valid method to use in determining differentiation success in iPSC-derived astrocytes.