Active Labeling with SmartBatch+

Reagents / Equipment Required:

• SmartBatch+

• SmartBatch+ Batch Single Sample Staining Kit or Batch Staining Kit

  • SmartBatch+ Primary Sample Buffer

  • SmartBatch+ Primary Device Buffer

  • Secondary Sample Buffer

  • SmartBatch+ Secondary Device Buffer

• Antibody Blocking Solution

• Donkey serum (any vendor will do)

• PBS with 0.02% sodium azide (PBSN)

• Labeling Reagents, such as primary and secondary antibodies or fluorescent nuclear dyes. Please consult Validated Antibody List for more information. 

• Staining Cup (Batch, Medium, or Single Sample sizes available)

• Mesh bag inserts

• Ring stand

• Lock Ring

• Sample cup storage solution. More can be made fresh here.

Refer to the timeline above. The red dots indicate optional stopping points. If you are not ready to move on to the next steps, move the samples to PBSN at 4°C until you are ready to proceed. 

Choosing appropriate antibodies and dyes

The protocol can accommodate:

• staining with dye-conjugated antibodies

• simultaneous delivery of primary along with monovalent Fab fragment secondaries

• sequential delivery of primary and secondary (can be Fab or IgG)

Please consult the Validated Antibody List for more information as it can take significant time and work to validate and optimize antibodies. We have also found that some antibodies require specific secondary delivery schemes, or are only bright enough to work in certain channels.

Are you using an antibody for the first time?

We recommend first following the Antibody Validation Protocol. If you wish to skip that step, we recommend using a sequential delivery of primary and secondary for the first test as simultaneous Fab fragments can alter the primary antibody binding affinity. It is also recommended to try new antibodies with 1-2 samples first, as batch experiments can require large amounts of antibody. 

Additional notes and recommendations:

• If you anticipate a signal being dim, we recommend sequential delivery of secondary using whole IgG secondaries in the 647 nm channel. 

• Avoid antibody host conflicts with primary antibodies.

• Avoid excitation/emission wavelength conflicts between secondary antibodies.

•  If you are delivering a nuclear dye or vasculature stain, please add them during the primary step. In general, we recommend using longer wavelengths (red or far red) for antibodies and saving shorter wavelengths for brighter targets or nuclear dyes.

• If you are using a dye, do so during primary labeling. To prevent the cup from being contaminated with the dye, use the same cup during secondary staining and post wash. This pulls any dye that got stuck in the cup during primary labeling back out of the cup. 

  • If you do not use the same cup in secondary labeling, quarantine the cup to use only with other samples being stained with the same dye. 

PRIMARY WASHING & PFA FIXING 

Day 3

1. Check to see if the experiment is complete.

• Open the Chamber lid. Use a pipette to remove ~20 µL of solution from the Sample Cup and pipette it onto a pH paper strip. 

• If the pH is still above ~8.3, you will need to extend the duration of the experiment to complete antibody binding. 

  • For every 0.1 above 8.3, the experiment should run for an additional hour. 

    • E.g. if you measure a pH of 8.5, change the timer to 2:00 (2 hours) and turn on Electrophoresis Power and Timed Shutdown.

• When the pH has dropped to 8.3 or below and labeling is complete, open the device and remove the Sample Cup. 

2. Move the samples to PBSN.

   • • Samples can remain in their Mesh Bags.

     • Batch labeled samples can remain on the Lock Ring.

For batch or medium sample experiments:

• Prepare an Incubation Jar with ~250 mL of PBSN.

• Remove the Lock Ring from the Sample Cup and place it on a Ring Stand. Place the Ring Stand in the Incubation Jar and close the lid. 

For single sample experiments:

• Prepare a conical tube with ~40 mL of PBSN.

• Remove the Mesh Bag with the sample from the Sample Cup and place it in the tube. 

Wash the sample until the end of the day in PBSN at RT with light shaking, refreshing the solution at least once. If your sample contains fluorescence, protect from light. 

3. Clean the device and cup.

• Carefully rinse the Sample Cup with distilled water and store it in its storage solution. It is important to keep the membrane hydrated at all times. We recommend refreshing the storage solution every few months. More can be made here. 

• Wash the device before shutting it down.  

4. PFA fixation.

[Note] Whether or not you add sequential secondaries, we recommend PFA fixing the samples to prevent antibody dissociation. If you are not ready to do an overnight PFA fix, simply keep the samples in PBSN at 4°C until you are ready. Extended time before fixing can result in antibody dissociation from epitope binding sites.  

• Samples can remain in their Mesh Bags.

• Batch labeled samples can remain on the Lock Ring.

• At the end of the day, prepare a solution of 4% PFA in 1X PBS. 

For batch staining:

• Fill an Incubation Jar with ~250 mL of 4% PFA.

• Place the Lock Ring on a Ring Stand and add to the Jar.

For single samples:

• Fill a fresh conical tube with ~20 mL of 4% PFA and add the sample. 

Incubate samples overnight at RT with light shaking protected from light. 

Sequential Secondary Labeling

Day 4

If you are ready to complete secondary staining in this day (immediately after PFA fixation), you can immediately continue to the next step. Otherwise, move the sample to PBSN and store it at 4°C protected from light until you are ready. 

1. The next morning start Secondary incubation at 37°C.

[Note] You do not need to PBSN wash between PFA and secondary sample buffer because the buffer contains tris which helps neutralize the PFA.

• For batch staining: wash out the Incubation Jar and replace the liquid with ~250 mL of Secondary Sample Buffer. Move the samples to this jar. 

• For single samples: move the sample to a fresh tube of ~20 mL of Secondary Sample Buffer.

Incubate samples for 6-8 hours at 37°C with light shaking, refreshing the solution once halfway through the incubation.

2. Around 4 pm, prepare the cup and device for secondary labeling.

Cup preparation:

• Wash and leak test the correct size sample cup as done during primary labeling.

• Choose the correct cup based on how many samples you have.

Device Preparation:

• Wash and dry the device

• Ensure the drainage valve is closed and add 300 mL of SmartBatch+ Secondary Device Buffer into the Chamber. This is the contents of an entire bottle.
  [Note] It is normal for this solution to be cloudy. It contains an insoluble defoamer to prevent excessive bubble buildup. 

3. Prepare samples and solutions.

Load the sample buffer and samples into the cup.

• In a leak-tested cup, pour a small amount of Secondary Sample Buffer into the cup and swirl it around to coat the membrane. Dump and discard the liquid in the cup. Repeat this a second time. 

• Fill the sample cup with Secondary Sample Buffer.

• Single Sample Staining Cup: 9 mL

• Medium Staining Cup: 20 mL

  • Batch Staining Cup: 40 mL 

Add secondary antibodies and dyes as needed to the Sample cup.

• Check the validated antibody page to see what molar ratio between primaries and secondaries you should be using. 

  • For most but not all antibodies, 2:1 is the molar ratio recommended for Secondary:Primary. Check the Validated Antibody list for more information.

• Add Normal Serum (Optional).

  • Use Normal Donkey Serum if you will use donkey host secondaries, and Normal Goat Serum for goat host secondaries.  

    1. • Single Sample Staining Cup: 200 µL

       • Medium Staining Cup: 1200 µL

       • Batch Staining Cup: 2400 µL

Load the samples into the cup.

• If you have not already, place each sample in a mesh bag, keeping track of which samples is where.

  • One mesh bag can hold one brain.

  • If you are using a medium or batch cup and have not loaded the samples onto the lock ring, do so now.

4. Load the cup into the device and start the experiment.

• Place the sample cup into the Chamber. Line up the hex on the bottom of the cup with the hex in the Chamber. 

• Turn on the Auxiliary Power while the lid is open and ensure that the sample cup rotates and is mixing well. 

• Close the Chamber Lid and Case Lid.

• Press the “Preset” button until the Labeling 2 preset is highlighted. 

  • Check the settings.

  • 30°C, 90V, and 400 mA current limit. 

  • Change the timer to 12 hours.

• Turn on Electrophoresis and Timed Shutdown. 

  • The timer will automatically turn off electrophoresis at the end of the experiment.  

  • These are normal starting values for current and voltage (They can vary more depending on temperature):

    1. Current: 400mA

    2. Voltage: 55-65V

Post Secondary Electrophoretic Wash and PFA Fixing

Day 5

1. When the  secondary labeling experiment is over, start the post secondary electrophoretic wash.

[Note] There will be no pH drop during secondary labeling, so there is no need to check the pH.

[Note] There is no need to replace the secondary device buffer for the post wash.

• Open the device and remove the Sample Cup. 

• Remove the samples from the cup.

• Discard the antibody buffer cocktail from the sample cup.

• Fill the cup with a small amount of Secondary Sample Buffer, swirl gently and discard. Repeat a second time. 

• Fill the cup with the appropriate amount of Secondary Sample Buffer depending on cup size and place the samples back into the cup.

• Place the sample cup back into the device, lining up the hex pieces.

• Turn on the Auxiliary Power while the lid is open and ensure that the sample cup rotates and is mixing well. 

• Change the timer to 3:00 (3 hours).

• Turn on electrophoresis and start the timer. 

3 hours later, repeat step 1 and allow the device to run for 3 more hours. In total, this is a 6 hour wash, with a refresh halfway.

2. Move the samples to PBSN.

For batch labeling:

• Wash out an Incubation Jar and fill it with ~250 mL of PBSN.

• Remove the Lock Ring from the Sample Cup and place it on a Ring Stand. Place the Ring Stand in the Incubation Jar and close the lid. 

For single sample experiments:

• Prepare a conical tube with ~40 mL of PBSN.

• Remove the Mesh Bag from the Sample Cup and place it in the tube. 

Wash the sample until the end of the day in PBSN at RT with light shaking, refreshing the solution at least once. If your sample contains fluorescence, protect from light. 

3. Clean the device and cup.

Carefully rinse the Sample Cup with distilled water and store it in its storage solution. It is important to keep the membrane hydrated at all times. We recommend refreshing the storage solution every few months. More can be made here. 

• Wash and shut down the device.

4. PFA fixation.

[Note] The secondary antibodies will now be fixed at the end of the day. If you are not ready to do an overnight PFA fix, simply keep the samples in PBSN at 4°C until you are ready. Extended time before fixing can result in antibody dissociation from epitope binding sites.  

• At the end of the day, prepare a solution of 4% PFA in 1X PBS. 

For batch staining:

• You will need ~250 mL of 4% PFA.

• Remove the Ring Stand from the Incubation Jar and wash out the Jar. Then fill it with the 4% PFA and insert the Ring Stand. 

For single samples:

• You will need 20 mL of 4% PFA. 

• Move the sample from PBSN to a fresh tube of 4% PFA. The samples can remain in their mesh bags. 

Incubate samples overnight at RT with light shaking protected from light. 

Day 6

1. The next morning transfer the samples to 40 mL of PBSN to wash out the PFA. 

   • Wash for at least 6 hours, refreshing the PBSN once halfway through.

You have completed Immunolabeling. Move onto Index Matching.