Removing Background

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vilsy
vilsy's picture
Removing Background

Hi guys,
 
I have way too much backgroun in my staining. Can you suggest me a way to remove it.
I use xylene based DPX mounting medium. Do you think it can be the reason for the background as opposed to using Vectashield mounting medium?
 
Thanks,
Vilay

Arvind Singh Pundir
Arvind Singh Pundir's picture
vilsy wrote:

vilsy wrote:

Hi guys,
 
I have way too much backgroun in my staining. Can you suggest me a way to remove it.
I use xylene based DPX mounting medium. Do you think it can be the reason for the background as opposed to using Vectashield mounting medium?
 
Thanks,
Vilay

Dear Vilsy
DPX and vectashield are used under different conditions as DPX is used generally for bright field and is not soluble in water as its a organic solvent and vectashield is used to Prevent rapid loss of fluorescence(photobleaching) while using rhodamine,texas red,fluorescein and other fluorochromes and its an aqueous mounting media so there is no comparison of both the mounting media , in case of both the mounting media they have nothig to do with the background staining, it may be because of other factors in both the cases  i.e, bright field(DPX) and fluorescence(vectashield mounting medium) like lack of washing in between the steps with suitable buffers , too much or too low dilution of your primary and secondary antibody,due to lack of blocking etc., so please elaborate your querry so that we can find out the culprit step and diagnose

samm
samm's picture
vilsy wrote:

vilsy wrote:

Hi guys,
 
I have way too much backgroun in my staining. Can you suggest me a way to remove it.
I use xylene based DPX mounting medium. Do you think it can be the reason for the background as opposed to using Vectashield mounting medium?
 
Thanks,
Vilay

 
Since you are mentioning DPX and Vectashield in the same sentence, I presume you are performing HRP staining. Are you performing the peroxide incubation step prior to adding primary Ab? Many cells have intrinsic peroxidase activity - unless that is neutralized with saturated peroxide, you'll see some bkground from it. Ensure that your blocking steps are adequete (based on sample, primary/sec/tert rgts), and use something like the universal serum block in the Vectastain Elite kit.
Btw, wrt Pundir's post, Vectashield is NOT the most ideal mounting medium for fluorescence either - even the DP-1000 series. Stuff like ProLong Xr works much better for critical samples (also costs more and can be a pain to use, so you need to balance it out). If you are doing IF as opposed to IHC, try dipping the slides in ice-cold methanol for 10-15 seconds prior to blocking and primary Ab addition -this reduces cellular autofl in many tissues and cells, esp in the green channel. Finally, use red-shifted dyes whenever possible.

vilsy
vilsy's picture
Thanks guys for your reply. I

Thanks guys for your reply. I am explaining what I am doing if you think it might help:
I am working with mice brain.
blocking solution : NGS (20%) in PBS
Primary Antibody 1:  Mouse-anti-NeuN conjugated with Alexa488  (so no secondary needed)
Primary Antibody 2:  Rabbit-anti-GAD67
Secondary antibod2 2 : goat-anti-rabbit with Rhodamine
 
Here is the protocol:
1. Animals were perfused with ice cold PBS followed by 4% PFA. Brian removed and postfixed in 4% PFA for 3 days
2. Soaked in 30% Sucrose solution till it sinks to the bottom
3. Frozen in OCT and sectioned in cryostat at 30um and mounted on subbed slides and stored at -20C
4. Slides were transferred to 4C for 30 mins and then RT for 30 mins (in a hood)
5. Slides are then washed in distilled water for 30s and air dired in the hood at RT for 20 mins
6. Sections were then washed in 1X PBS, 3 times for 10 mins each
7. Once in PBST (0.3% Triton X)
8. Three washes in PBS
9. Incubation with blocking serum (20% NGS in PBS) for 1 hr at RT
10. Primary antibodies were diluted in PBST and then incubated overnight at 4C
11. Washed thrice with PBS (for NeuN where no secondary needed) or PBST (for GAD67 where secondary is needed)
12. Secondary incubation for 2 hrs at RT  with dilution done in PBST
13. Three washed with PBS followed by three washes with distilled water each for 10 mins each
15. slides are then transferred to a 20% EtOH solution for 5 mins, then 50% EtOH for 7 mins, then 70% EtOH for 7 mins, then 95% EtOH for 15 mins and 100% EtOH for 20 mins
16. Transferred to Xylene for 15 mins
17. Slides are allowed to air dry and xylene to evaporate then mounted with DPX
 
My problem is too much background and non-specific staining. I have tried it three times and have the same problem consitently.
 
Thanks,
Vilay

Arvind Singh Pundir
Arvind Singh Pundir's picture
Dear Vilsy,

Dear Vilsy,
as you are using secondary antibody tagged to rhodamine and alexa fluor , then why are you using alcohol, xylene and DPX step as your are performing IF you should first use anti fading mounting media , rhodamine reacts with alcohol and will fade your signal try using aqueous antifading mounting media instead of DPX and also edit the alcohol and kylene step in your 13th, step after three washes with PBS mount the slide with mounting media any glycerol based mounting media or you can use vectashield it works good, secondly try reducing your serum concentration in blocking step from 20% to 5 or 10% hope this works for you......good luck

samm
samm's picture
Hi Vilsy! I've added some

Hi Vilsy! I've added some notes to your protocol below - these are things you might want to try optimizing.
Best
-sam
pundir wrote:

vilsy wrote:
Thanks guys for your reply. I am explaining what I am doing if you think it might help:
I am working with mice brain.
Since you have a mouse Ab and mouse tissues thats an added concern: have you tried the Vector Labs Mouse-On-Mouse (MOM) kit?
blocking solution : NGS (20%) in PBS Reduce serum to 5%
Primary Antibody 1:  Mouse-anti-NeuN conjugated with Alexa488  (so no secondary needed)
Primary Antibody 2:  Rabbit-anti-GAD67
Secondary antibod2 2 : goat-anti-rabbit with Rhodamine
 
Here is the protocol:
1. Animals were perfused with ice cold PBS followed by 4% PFA. Brian removed and postfixed in 4% PFA for 3 days
2. Soaked in 30% Sucrose solution till it sinks to the bottom
3. Frozen in OCT and sectioned in cryostat at 30um and mounted on subbed slides and stored at -20C
 
Have you tried using OCT/PBS directly on your tissue (placed in a cassette or foil boat), and evenly snap chilled (by holding it above a hexane solution in a metal beaker plaved in liqN2). PFA can give higher green background in some tissues.
 
7. Once in PBST (0.3% Triton X)
8. Three washes in PBS
Dip your slide in ice-cold Methanol (store it in -20dC prior to expt) for 10-15 secs. You may need to use a PAP pen again to mark section area on slide.
9. Incubation with blocking serum (20% NGS in PBS) for 1 hr at RT
Reduce serum to 5% - more does not help, and might hurt
10. Primary antibodies were diluted in PBST and then incubated overnight at 4C
Have you titred these for optimal signal:noise?
11. Washed thrice with PBS (for NeuN where no secondary needed) or PBST (for GAD67 where secondary is needed)
Only last wash should be with PBS - PBS-T improves specificity.
12. Secondary incubation for 2 hrs at RT  with dilution done in PBST
13. Three washed with PBS followed by three washes with distilled water each for 10 mins each
Only last wash should be with PBS - PBS-T improves specificity.
15. slides are then transferred to a 20% EtOH solution for 5 mins, then 50% EtOH for 7 mins, then 70% EtOH for 7 mins, then 95% EtOH for 15 mins and 100% EtOH for 20 mins
16. Transferred to Xylene for 15 mins
Skip steps 15 and 16 above (and 17 too). Quickly dip slides in water 2x to remove PBS (3x10 mins not req), blot around section on slide using kimwipes, add mounting medium.

 Use Vectashield (f1000 series, Vector Labs), or Fluorsave (Calbiochem) or Prolong - or any other similar product with anti-fade, that lists your flours as approved.
My problem is too much background and non-specific staining. I have tried it three times and have the same problem consitently.
 PBS-T washes, methanol treatment and correct dilutions should greatly improve specificity.
 

Dear Vilsy,
as you are using secondary antibody tagged to rhodamine and alexa fluor , then why are you using alcohol, xylene and DPX step as your are performing IF you should first use anti fading mounting media , rhodamine reacts with alcohol and will fade your signal try using aqueous antifading mounting media instead of DPX and also edit the alcohol and kylene step in your 13th, step after three washes with PBS mount the slide with mounting media any glycerol based mounting media or you can use vectashield it works good, secondly try reducing your serum concentration in blocking step from 20% to 5 or 10% hope this works for you......good luck

heehawmcduff
heehawmcduff's picture
As mentioned previously I

As mentioned previously I would endorse a thorough wash after your final labelling step and prior to mounting your samples in mounting medium - it's amazing how much the background can be reduced from such a simple procedure.
Regards

vilsy
vilsy's picture
Thanks guys. Will try these

Thanks guys. Will try these changes and let you know how it goes!

vilsy
vilsy's picture
Hi Guys,

Hi Guys,
I tried the suggested changes. i must say that the quality of ny NeuN staining has really improved.
 
Thanks,
Vilay