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Possible causes i can think of:
1) less washing
2) high Abs concentration
Steps taken to solve the problem:
1) Increased washing (in duration only, not in terms of force applied, as i wash with PBS poured from syringe. I increased the total no of times of washing as well as total time during the washing)
2) for one FROZEN cryosection (10µm), i am using 200µl of solution. In this solution, laminin concentration is 1:200. The secondary Ab i am using is Rohdamine with concentration = 1:400.
I decrease the rohdamine concentration to 1:600 and 1:800, but still have the back ground.
Please reply,
Thanks
Best,
AR
Mon Aug 15, 2011 5:52 am
gula
Rank: Member
Joined: Mar 20, 2008
Posts: 94
Post subject:
Comparing your picture to others found in google I think your are not far away from a beautiful stain.
I would try to decrease the primary antibody and test 1:400, 1:600 with the secondary at 1:800.
Go down with the concentration until you think your results are too faint.
I don't know your special stain, but sometimes the publicated images are a little bit manipulated to get better contrast or diminish background. ; )
I can imagine, that especially in a dense structure like muscle a certain degree of background cannot be fully prohibited.
Thanks Gula for the reply,
I will follow your advice in my today's experiment and will report the outcomes.
Best,
AR
Mon Aug 15, 2011 1:10 pm
AR
Rank: Member
Joined: Jun 10, 2011
Posts: 59
Post subject:
Hi,
I have a feeling, apart from high Abs concentration and/or less washing, that freezing artifact (wavy muscle fibers outlines) can lead to laminin background staining (in-spite of decreasing Abs concentration and increasing washing). I noticed laminin background more with areas where freezing artifacts are present.
Please clarify!
Best,
AR
Mon Aug 15, 2011 1:19 pm
AR
Rank: Member
Joined: Jun 10, 2011
Posts: 59
Post subject:
Hi Gula!
Thanks for the advice. It worked for me!
With laminin 1:600 and rhoodamine 1:800 the background was very less (decreased by ~60%); however still some background is present.
Do you think that reducing the Ab concentration further might help.
Should i reduce both the laminin and rhodammine concentration (say laminin 1:800 and rhodamine 1:1000).
I am just concern that such low concentration might negatively affect the staining.
Need your experience with this.
Hope to hear from you soon.
Thanks
Best
AR
Wed Aug 17, 2011 4:37 am
Carl
Rank: Member
Joined: Oct 11, 2004
Posts: 1871
Post subject: Well done, AR....
...and Gula!
Why not do primary at 1/500, 750, 1000 and for each of those dilutions, do 3 secondary dilutions of 1/500, 750, 1000.
So, 9 sections total.
Once you have seen which secondary gives the best sig:noise, you will never need to alter the concentration/dilution factor of the secondary.
It will then only be your new primary Abs that you will have to titrate.
Remember, the best titration range is one where the staining is too strong at the most concentrated and too weak at the most dilute.
That is the only way to quickly find the optimal dilution factor for a primary Ab.
Wed Aug 17, 2011 1:17 pm
Carl
Rank: Member
Joined: Oct 11, 2004
Posts: 1871
Post subject: Well done, AR....
...and Gula!
Why not do primary at 1/500, 750, 1000 and for each of those dilutions, do 3 secondary dilutions of 1/500, 750, 1000.
So, 9 sections total.
Once you have seen which secondary gives the best sig:noise, you will never need to alter the concentration/dilution factor of the secondary.
It will then only be your new primary Abs that you will have to titrate.
Remember, the best titration range is one where the staining is too strong at the most concentrated and too weak at the most dilute.
That is the only way to quickly find the optimal dilution factor for a primary Ab.
Wed Aug 17, 2011 1:17 pm
AR
Rank: Member
Joined: Jun 10, 2011
Posts: 59
Post subject:
Carl !
I will follow your advice also today itself.
Thanks!
Wed Aug 17, 2011 3:09 pm
gula
Rank: Member
Joined: Mar 20, 2008
Posts: 94
Post subject:
Hi Carl!
Making a titration-array is certainly the most elegant way.
My thought was, that AR isn't far away from an optimal result, so the change of only one player was my option.
I also thought, that a high diluted primary combined with a less diluted secondary has more chance to get a clear stain than the other way round.
But that could be just "woodoo" ; )
gula
Wed Aug 17, 2011 6:30 pm
AR
Rank: Member
Joined: Jun 10, 2011
Posts: 59
Post subject:
Carl!
Just done with following your advice also. The staining has improved considerably with titer: primary 1/1000 and secondary 1/1000. Little red haziness in the background still persists.
I have attache the sample image at
http://www.immunoportal.com/modules.php?name=gallery2&g2_itemId=18019&
Few things:
1) the established protocol followed in my lab use laminin 1/200 and rhodamine 1/400. I am feeling a little uneasy regarding the concentration being too low (1/1000). Do you think its too low? In addition, i have increased both the no of steps and duration of washing than before.
2) Now only primary Ab concentration needs to be manipulated. Should i go down more from 1/1000?
Thanks
Best,
AR
Thu Aug 18, 2011 11:40 pm
Carl
Rank: Member
Joined: Oct 11, 2004
Posts: 1871
Post subject: You are right, Gula
Respect.
It was just that it did not seem as if AR had addressed the fact that for most purposes the detection reagent(s) should be initially optimised wrt dilution/concentration factor....and then there is no need, in general, to ever change this dilution factor, for any primary Ab, imho.
The only variable should be the dilution factor of the primary Ab.
So, AR is getting there!
A very nice result, AR.
Congratulations.
Sure....why not dilute further, just to see what you get.
NB:
1. Use Unsharp mask to sharpen the edges of the image
2. Set Black reference for the camera, if your software allows you this.
3. You have some ice-crystal artefact which may account for the slight background haziness.
Fri Aug 19, 2011 3:43 am
AR
Rank: Member
Joined: Jun 10, 2011
Posts: 59
Post subject:
Thanks Carl,
Just to be doubly sure:
1) i would just need to alter the primary Abs concentration now
2) presently the primary Ab i am using is 1/1000. At what factor should i start diluting , like 1/1200, 1/1400 (with an increase of 200µl) or what your experience say?
3) Also please clear my query:
- lowering down laminin stain concentration gives decent staining intensity, so it means the concentration i was using initially (1/200 for L and 1/400 for R) was too high.
- regarding ice crystal artifact: how come it can happen? clueless about temperature change causing this!
I take the section on slide> keep the slides in cryostat chamber for few minutes (~20 min)> take out the slide box and keep it at RT for 20min before start fixation and hence, immuno.
Fri Aug 19, 2011 3:43 pm
Carl
Rank: Member
Joined: Oct 11, 2004
Posts: 1871
Post subject: Hi AR
1. Yes but...also use another primary Ab that you know is good, just to check that the secondary dilution factor is good for all Abs ( it should be)
I prefer Alexa dyes and would use Alexa 594 in place of TRITC but, if you have lots of TRITC....keep using it until it runs out, because you are getting good results ( with laminin Ab....please tell me what anti Laminin Ab you are using)
2. I am sure that 1/1000 is very good so, just for your peace of mind, do as you suggest , increasing dilution factor by 200 until 1/2000.
Why?
Because you say that there is a slight background that you think that you can get rid of.
It may not be go away but....why not check it out, if you have time?
3. Yes. Some people think that if you increase concentration you will always get stronger staining..." (1/200 for L and 1/400 for R": I forget what L and R refers to
Every primary Ab should be titrated using a std concentration of secondary to show too strong staining at the lowest dilution factor ( thus the highest concentration of Ab) and no staining at the highest dilution factor. You get a bell curve of intensity so, you choose the dilution factor at the peak of the curve.
Thus, if you make slight errors, you still get near peak intensity.
If an Ab is applied at too high a concentration, you may get concentration inhibition, lots of background.
It also depends upon so many other factors which I do not understand.
It is empirical...so test and don't think about the theory until you get good results
Re ice-crystal artefact...hmmm, it is minimal ( sure, I may be wrong...anyone else please help...Gula?).
It is only/always due to freezing too slowly.
Yes....I freeze say 10 samples exactly the same way and 2 specimens show ice crystal artefact...grrrrr!
Perhaps you can also spend some time testing out variations on your freezing technique?
It will be worth it.
Anyway, I'm a Histologist so I tend to go on and on and on...
Sigh
Carl
Fri Aug 19, 2011 7:39 pm
AR
Rank: Member
Joined: Jun 10, 2011
Posts: 59
Post subject:
Hi Carl,
You and Gula have been such kind to me!
I will have to see the name of laminin Ab to just confirm once. I will go ahead and try lowering down laminin concentration with hodamine at 1/1000, and get beck here with some result. Hopefully good!
P.S: L = laminin, R = rhodamine
Best,
AR
Sat Aug 20, 2011 3:11 pm
AR
Rank: Member
Joined: Jun 10, 2011
Posts: 59
Post subject:
Carl!
I am totaled ..... I tried the lamining concentration manipulation with keeping the rhodamine concentration at 1/1000. I have not gotten favorable results. Almost all of the sections showed moderate background (as red haziness). The laminin i am using is = Rabbit polyclonal antibody. Please see the sample images at:
Few questions aside:
Q1 = Could the laminin background be due to freezing artifact? I did not see anything major in these section though, just wanted to ask.
Q2 = Could the laminin background be due to connective tissue/blood vessels? I too the image keeping the blood vessel as my landmark, but the background was more or less same in other parts of section.
Q3 = In few sections, the laminin staining was dull (compared to adjacent areas) in patchy areas. Does it means that the concentration of my primary AB has been decreased below the threshold level.
Q4 = Does secondary Ab need to be diluted further below 1/1000?
With your's and immunoportal's help, i have been able to cross lots of barriers in my immuno experiments, but still, i guess, i am not skilled enough
I donno what is happening with me
In dire need of guidance and ...support.....
Best,
AR
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