CST BLOG: Lab Expectations

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Subcellular Marker Selection for Immunofluorescence

When performing immunofluorescence (IF), it is common to multiplex with several antibodies and/or dyes to generate information about the protein of interest in relation to other markers. Of all the recommendation requests submitted to the the IF technical support team, the most popular markers mentioned are subcellular markers.

21-EMG-75937 Figure 0Subcellular markers are high-affinity reagents for organelle-specific components within a cell that can enable your research by providing a clear view of the physical and spatial characteristics of different organelles. There are many reasons to include a subcellular marker in your next immunofluorescence experiment – studying specialized organelle-specific functions, assessing drug effects through organelle health, or examining global changes in a knockout cell.  You may want to use a specific reagent for the protein of interest in your experiment and also want to include a reagent to determine subcellular localization. However, not all reagents are universally compatible. Reagents can perform differently depending on the model species and staining protocol. This guide has been designed to help you find the subcellular marker that fits your experimental needs. 

Experimental Design  

For the purpose of this experiment, we have focused on five major subcellular localizations: cytoskeleton, mitochondria, endoplasmic reticulum, golgi, and nuclear envelope.  

21-EMG-75937 Figure 1

There are several localizations which have been omitted here either because they have very specific markers (e.g. early endosomes with Rab5 or centrioles with PCM1)  or there are faster methods to label the localization (e.g. nuclear dyes like DAPI #4083 and Propidium Iodide #4087). 

21-EMG-75937 Figure 2

PCM-1 (G2000) Antibody #5213  

21-EMG-75937 Figure 3

Mouse retina stained with ProLong® Gold Antifade Reagent with DAPI #8961, Neurogranin #79519, and S6 #5548 

 

Only ubiquitous markers were considered in this analysis. Reagents evaluated in this experiment were tested in human, mouse, and rat cells using the three main workflows used for immunofluorescence experiments at Cell Signaling Technology® (formaldehyde fixation with Triton™ X-100 permeabilization, formaldehyde fixation with methanol permeabilization, and methanol fixation). 

 

With the goal of identifying the best marker for a given protocol or species, we have compared antibodies within a given localization assessing both protocol and species performance using two criteria: specificity and signal intensity. Performance across species and protocol have been normalized to the best performer within the localization for each class and evaluated on a scale of 0 - 100. This allows us to compare multiple variables in a radar chart (as shown in the plot below comparing beverage traits). 

 

21-EMG-75937 Figure 4

To avoid recommending products that give nonspecific patterns (e.g., nucleolar labeling with a mitochondrial marker) in a specific protocol or species, the product was given a 0 for the class it was evaluated in if nonspecific pattern was observed. 

 

For the purpose of this test, each antibody was tested in triplicate in a single well of a 96 well plate diluted at the recommended dilution in 100 uL of antibody diluent. Incubation of primary antibody was performed at 4°C overnight. Primary antibodies were labelled using host appropriate Alexa Fluor® 488 conjugated secondary (Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 or Anti-mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4408) antibody diluted at 1:500. Incubation of secondary antibodies were performed in the dark at room temperature for one hour. 96 well plates were analyzed by high content analysis including a laser scanning image cytometer to generate MFI data and an automated imager to generate 200X images of DAPI and the 488 channel.  

Results 

Comparing product performance throughout each subcellular localization using radar charts allowed us to identify the best performers for each category. While all radar charts are available at the end of this report, details on the best performers for each localization are featured here. 

Cytoskeletal Markers

When using cytoskeletal markers, the most experimental flexibility is gained by using a Phalloidin conjugate - a phallotoxin that tightly binds to filamentous actin. Because this is a non-antibody method of labeling, it allows you to conserve host species for other markers. Phalloidin has one downside for use - it is incompatible with methanol permeabilization or fixation.  

21-EMG-75937 Figure 5

If methanol is necessary in your experiments, α-Tubulin (DM1A) Mouse mAb #3873 provides robust signal in methanol treated samples.

21-EMG-75937 Figure 6

Keep in mind that tubulin labels a different component of the cytoskeleton (microtubules) compared to actin (microfilaments). The third component of the cytoskeleton - intermediate filaments - has different proteins depending on cell type and so it could not be included in this experiment. 

21-EMG-75937 Figure 7

Golgi Markers

We found that golgi markers favor formaldehyde fixation with Triton™ X-100 permeabilization. However, there is substantial protocol flexibility with GM130 (D6B1) XP® Rabbit mAb #12480 if you are working with human samples.  

 

21-EMG-75937 Figure 8

RCAS1 (D2B6N) XP® Rabbit mAb #12290 works in multiple species as long as formaldehyde fixation is included. 

 

21-EMG-75937 Figure 921-EMG-75937 Figure 10

Endoplasmic Reticulum (ER) Markers

We typically recommend testing formaldehyde fixation or formaldehyde fixation with methanol permeabilization when working with ER specific proteins. However, in this experiment, PDI (C81H6) Rabbit mAb #3501 worked well with all protocols and species tested. 

21-EMG-75937 Figure 11

Calreticulin (D3E6) XP® Rabbit mAb #12238 is incompatible with formaldehyde fixation alone and requires either methanol permeabilization or methanol fixation. 

21-EMG-75937 Figure 1221-EMG-75937 Figure 13

Mitochondrial Markers

There are many available markers for mitochondria. In house at Cell Signaling Technology®, the preferred mitochondrial marker is AIF (D39D2) XP® Rabbit mAb #5318 due to compatibility with many species and protocols. AIF Antibody #4642 slightly outperforms #5318 in terms of signal; however, we prefer XP® monoclonal antibodies in house.  

21-EMG-75937 Figure 14

In cases where methanol fixation is necessary and the species is human or rat, COX IV (3E11) Rabbit mAb #4850 may perform better than AIF antibodies.  

21-EMG-75937 Figure 15

When working with mouse cells, SDHA (D6J9M) XP® Rabbit mAb  #11998 will perform slightly better than AIF antibodies in methanol permeabilized sample. 

21-EMG-75937 Figure 1621-EMG-75937 Figure 17

Nuclear Envelope Markers

For nuclear envelope markers, there was a clear winner. Lamin A/C (4C11) Mouse mAb #4777 worked in all species and most protocols and gave clear bright signal which was slightly diminished with methanol fixation.

21-EMG-75937 Figure 1821-EMG-75937 Figure 19

Tips before You Begin

Above we have discussed some of the best performers for each subcellular localization. 

As always, the most important factor for experimental success is that samples are treated with care. Consider the health and confluence of your cells before fixation as these factors will impact your experiment. Make certain that your sample is sufficiently fixed and avoid allowing the sample to dry out as this can negatively affect an immunofluorescence experiment. 

21-EMG-75937 Table21-EMG-75937 Figure 2021-EMG-75937 Figure 2121-EMG-75937 Figure 22

21-EMG-75937 Figure 2321-EMG-75937 Figure 24

 

Ginny B.
Ginny B.
Ginny Bain is Group Leader of the Immunofluorescence Group at Cell Signaling Technology. A self-professed microscope nerd, Ginny also enjoys quantitative bioimaging, a nice t-SNE plot, and biking the north shore.

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