SmartBatch+ Secondary Labeling

Reagents / Equipment Required:

• SmartBatch+

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

  • 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.

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:

• If your device is upgraded for Radiant Labeling, make sure to remove the Radiant Column from the device!

• 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.

  • Look at the samples inside the cup from the side through the membrane. If you see any bubbles between the bag and the sample, you can lower a pipette or small spatula into the cup to remove the bubble. 

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.