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    • CommentAuthorjuruble
    • CommentTimeJun 15th 2007 edited
     

    This protocol explains how to: immunostain cultures with 3A10 to identify retinal ganglion cells.

    Protocol revised: 6/15/2007

    Protocol written by: Barbara Lom

     

    I. Make PBT Solution

    PBT is a solution of 1X phosphate buffered saline (PBS) with 0.2% bovine serum albumin (BSA) and 0.1% Triton-X. PBT cannot be stored longer than one day and needs to be made fresh. Make up at least 100 ml, but do not make much more than you need.  You can mix this solution in a 250 ml glass jar.

    • If you are doing all steps in immunostaining in one day you’ll need ~35 ml PBT/dish
    • If you are only doing the primary antibody today you’ll need ~15 ml PBT/dish today (and ~20 ml PBT/dish tomorrow)


    For example, for 100 ml of PBT you would mix:

    0.2 g BSA (bovine serum albumin fraction V)
    100 ul Triton-X (a detergent)
    ~99 ml PBS (to bring solution up to 100 ml)


    II. Prepatory Rinses

    When adding liquid to culture dishes, use a plastic pipette and add it slowly against the side of the dish to minimize the flow over the coverslip.

     


    Video of carefully removing and adding liquid to culture dish. 



    A. Gently remove most (but not all) of the liquid from your culture dish and discard it in a waste container.
    Gently add ~2.5 ml of PBT and shake on the horizontal shaker set at a low speed for ~5 minutes.

    B. Gently remove most (but not all) of the PBT from your culture dish and discard it in a waste container.
    Gently add ~2.5 ml of PBT and shake on the horizontal shaker set at a low speed for ~5 minutes.

    C. Gently remove most (but not all) of the PBT from your culture dish and discard it in a waste container.
    Gently add ~2.5 ml of PBT and shake on the horizontal shaker set at a low speed for ~5 minutes.

    D. Gently remove most (but not all) of the PBT from your culture dish and discard it in a waste container.
    Gently add ~2.5 ml of PBT and shake on the horizontal shaker set at a low speed for ~20 minutes.


    III. Blocking Solution


    A. During the long rinse make your blocking solution. Calculate how much blocking solution you will need for the day and only make as much as you will need. You can mix this solution in a 50 ml sterile centrifuge tube.  Blocking solution is 5% goat serum in PBT.

    For 10 ml of blocking solution you would mix:

    500 ul goat serum
    9.5 ml PBT

    B. Gently remove most (but not all) of the PBT from your culture dish and discard it in a waste container
    Gently add ~2.5 ml of blocking solution and shake on the horizontal shaker set at a low speed for ~20 minutes.


    IV. Primary Antibody

    A. During the blocking solution rinse make your primary antibody solution. Use the 3A10 primary antibody at a 1:100 (a.k.a. 1% v/v) dilution. Make only as much as you need (antibodies are VERY expensive reagents). You can mix this in a 1.5 ul microfuge tube.

    For 250 ul of primary antibody solution you would mix:

    2.5 ul 3A10 antibody
    247.5 ul blocking solution

    B. Gently remove most (but not all) of the blocking solution from your culture dish and discard it in a waste container. Gently add 250 ul of primary antibody solution.

    C. If you want to do all your immunostaining in one day: Shake your dish low speed for at least 3 hours. If you want to finish your immunostaining tomorrow: Secure the lid with parafilm and store in the fix frig overnight.



    V. Rinsing off the Primary Antibody


    A. Add ~2.5 ml PBT to your culture dish and then gently remove most (but not all) of the liquid from the dish and discard it in a waste container. Gently add ~2.5 ml of PBT and shake on the horizontal shaker set at a low speed for ~5 minutes.

    B. Gently remove most (but not all) of the PBT from your culture dish and discard it in a waste container. Gently add ~2.5 ml of PBT and shake on the horizontal shaker set at a low speed for ~5 minutes.

    C. Gently remove most (but not all) of the PBT from your culture dish and discard it in a waste container. Gently add ~2.5 ml of PBT and shake on the horizontal shaker set at a low speed for ~5 minutes.

    D. Gently remove most (but not all) of the PBT from your culture dish and discard it in a waste container. Gently add ~2.5 ml of PBT and shake on the horizontal shaker set at a low speed for ~20 minutes.


    Use foil &/or a slide box to protect 2° Ab from light for all remaining steps


    VI. Secondary Antibody


    A. During the last PBT rinse solution rinse make your secondary antibody solution. Use the goat anti-mouse Alexa 488 secondary antibody at a 1:500 (a.k.a. 0.2% v/v) dilution. Make only as much as you need (antibodies are VERY expensive reagents).
    You can mix this in a 1.5 ul microfuge tube.

    For 250 ul of secondary antibody solution you would mix:

    0.5 ul Alexa 488 antibody (use the P2 pipette)
    249.5 ul blocking solution

    B. Gently remove most (but not all) of the blocking solution from your culture dish and discard it in a waste container. Gently add 100 ul of secondary antibody solution and cover very gently with a circle of parafilm.

    C. Shake your dish low speed for ~1.5 hours (make sure the antibody is protected from light with foil or a box). The secondary antibodies will adhere nonspecifically if left on the slides for too long.


    VII. Rinsing off the Secondary Antibody


    A.
    Add ~2.5 ml PBS to your culture dish and then gently remove most (but not all) of the liquid from the dish and discard it in a waste container. Gently add ~2.5 ml of PBS (note: PBS not PBT here) and shake on the horizontal shaker set at a low speed for ~5 minutes.

    B. Gently remove most (but not all) of the PBS from your culture dish and discard it in a waste container.
    Gently add ~2.5 ml of PBS and shake on the horizontal shaker set at a low speed for ~5 minutes.

    C. Gently remove most (but not all) of the PBS from your culture dish and discard it in a waste container.
    Gently add ~2.5 ml of PBS and shake on the horizontal shaker set at a low speed for ~5 minutes.

    D. Gently remove most (but not all) of the PBS from your culture dish and discard it in a waste container.
    Gently add ~2.5 ml of PB (note: PB not PBS here) and shake on the horizontal shaker set at a low speed for ~20 minutes.


    VIII. Finishing/Mounting

    Place a drop of gelmount on a microscope slide. Gently lift coverslip out of culture dish using fix forceps. Lean the edge of the slide against a kimwipe to soak off some moisture. Gently “roll” coverslip face down onto the gelmount, being careful not to slide it once it touches down. Seal the edges with nail polish. Store in a slidebox in the fix frig. Take images within a day or two of immunostaining for best results.

     

    Example of immunostained RGC:

     

    Xenopus laevis retinal ganglion cell immunostained with 3A10

     

    • CommentAuthorredsox07
    • CommentTimeAug 5th 2007 edited
     

    If you want to stain with phalloidin as well as the 3A10 antibody, here is how we did it during the summer of 2007:

    Add 5uL of phalloidin to each plate after the plates have been treated with the secondary antibody for 1 hour.  Since the secondary antibody is designed to stay on for about 1.5 hours and the phalloidin for about .5 hours, this will syncronize when you can wash off both stains.

    Also, we tried using different concentrations of phalloidin instead of just the 5uL that was suggested.  We tried 1uL, 2.5uL, and 5uL on a sample experiment, but never completely analyzed the experiment to the end because it was just a trial.  I would suggest that if you are not satisfied with the stain that you are getting with 5uL, try increasing and decreasing the concentration slightly to find a better way.

    We just started using phalloidin in the summer of 2007, so we have not really found the perfect protocol for it yet.  If you are not satisfied with the way that it stains after you have imaged and measured your neurons, I would suggest talking to Dr. Lom and Dr. Watson about ways that you could improve the stain, either by leaving it on for a longer time or by messing with the concentration.  I found that when I was imaging the phalloidin stained neurons using red fluorescence, it did not appear that I was getting very good pictures, but as soon as I started measuring neurites using Image-Pro Plus, I found that the different builds (especially red screened on top of green) were very helpful and allowed me to see things that I couldn't with just phase or the other fluorescence.  So I would not give up on phalloidin just yet if it appears that you are not satisfied with the results, but I encourage you to tweak the protocol to try to improve the staining procedure as best you can.

    Also, upon first obtaining a new bottle of phalloidin, be sure to dissolve it in methanol as per the instructions that come with the phalloidin.  However, we have already done this with the phalloidin that was received during the summer of 2007.

    Mike, summer 2007

    • CommentAuthorredsox07
    • CommentTimeAug 5th 2007 edited
     

    I just re-read the above protocol and it says to leave the secondary antibody on for only ~1 hour, not 1.5 as I had stated above.  However, in the protocol I receieved from Dr. Watson, 1 is crossed out and 1.5 is written in, and I think that we used 1.5 during my experiments and it turned out pretty well.  So consult with someone about the time to leave the secondary antibody on, and then add the phalloidin when there is 30 minutes left for the secondary antibody to stay on.  Also, I looked at my notes and we ended up leaving the phalloidin on for a little longer than just 30 minutes (more like 35-40 minutes), so obviously there is some flexibility in the timing of the stains.

    Mike, summer 2007

    • CommentAuthorkilang
    • CommentTimeNov 11th 2007
     
    Making PBT:
    If you add the PBS to the container first (before the Triton-X), you can pipette the PBS up and down to help get a bit more Triton-X out of the pipette tip (the detergent is really thick).

    Primary Antibody:
    I had much more success when I left the primary antibody on overnight (after letting the dish shake for a few hours) than when I only left it on for the 3 hours suggested above.
    • CommentAuthorkacole
    • CommentTimeNov 11th 2007
     
    In order to maxmize fluoresence, you can double the concentration of the primary antibody. I let the 1*Ab rock for >4 hours and incubate overnight before rinsing and adding the 2*Ab to ensure maximum binding.

    Also, you can let the coverslip sit in the final PB rinse overnight before coverslipping if you don't have time after the final wash.

    One more tip: whenever mixing solutions, always add the solution requiring the most volume first so that you can use the volume to add the other components, aspirating up and down with the pipette tip several times to make sure you add all of the component and none is stuck inside the tip.
    • CommentAuthorjuruble
    • CommentTimeJan 4th 2008 edited
     

    Originally posted by Courtney Cron. This comment will help you schedule your immunostaining:

     

    *The entire immunostaing procedure takes approximately six hours and forty-five minutes (06:45:00). Be sure to allow at least this much time if you plan to complete immunostaining in one day. Below is the break down of time for each step, indicating when to split the procedure into a second day if necessary:

    I. 10 minutes - Make PBT Solution
    II. 35 minutes - Preparatory Rinses
    III. 20 minutes - Blocking Solution
    IV. 3 hours - Primary Antibody
    *V. 35 minutes - Rinsing off Primary Antibody  (FLEXIBLE STEP: You may do this on Day 1 or wait until Day 2, please see section heading for particulars)
    VI. 1.5 hours - Secondary Antibody
    VII. 35 minutes Rinsing off Secondary Antibody
    VIII. Mounting only takes a few minutes and can be done anytime within 24 hours of immunostaining

    • CommentAuthorjuruble
    • CommentTimeMar 4th 2008 edited
     

    Dom Ippolito created an immunostaining checklist that is sized to adhere in a 9.5" x 6" lab notebook.  Download the following Excel file and print it out to use this checklist, which will help you keep track of what immunostaining steps you have completed.

     

     

     

    Example of spreadsheet being used to manage immunostaining.

     

    • CommentAuthoraldeal
    • CommentTimeJun 21st 2010
     
    WHEN MAKING BLOCKING SOLUTION: Note that when making blocking solution it is used in the primary and secondary antibody solutions as well as the 20 min blocking rinse.
    This is the list of total blocking solution that should be made:
    2.5 ml per dish (blocking rinse)
    247.5 ul per dish (primary antibody)
    If you are doing the entire process in one day also include:
    99.8 ul per dish (secondary antibody)
    • CommentAuthoraldeal
    • CommentTimeMay 18th 2011 edited
     

    I have made a new immunostaining checklist that helps me obtain the proper calculations and measurements. If you are not careful, you may not make enough PBT or blocking solution for later solutions. This new checklist better accounts for later solutions. This checklist is sized to fit in a 9.5" x 6" notebook.

     

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  1.  

      *****ORIGINALLY BY ALEX DEAL. I think it is nice to have it in this protocol as well******

     

     

    This protocol explains how to: remove coverslip from the bottom of a poly-o movie dish

    Protocol revised: 6/18/2010

    Protocol written by: Alex Deal

    This protocol was obtained from the MatTek Corporation website (http://www.glass-bottom-dishes.com/faq.html) under section A-DU5:

    1. Order Part # PDCF OS 30 (Fluid for removal of coverslips from glass bottom dishes)

    2. Invert the cover of the dish.

    3. Pipette 1.0 ml of fluid into the inverted cover.

    4. Place the bottom of the dish in the cover. Make sure that the liquid is touching the bottom of the coverslip.

    5. Allow the dish to sit in the fluid for 45 minutes at room temperature.

    6. Dry the bottom of the coverslip with an absorbent paper towel.

    7. Place the dish on a clean surface. Using forceps, press down on the edge of the coverslip to separate the coverslip from the dish.

    Note: If the above procedure is followed, the PDCF OS 30 fluid will not contact the cells and will not disrupt cells on the coverslip or the staining thereof.