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Hi

One concern I have, is it known if the caffeine is some how bound in 
this material, as caffeine is freely soluble in water in its pure form.

Other than that I normally use neat serum from the same species as the 
2nd antibody as the blocking protein.


Russ

On 10/13/2010 9:25 AM, Shane van Breda wrote:
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>
> Dear list server members,
>
> I am busy with my MSc and a while back I posted a question about localising
> caffeine in tea (Camellia sinensis) leaves. We have made progress with normal
> stains but I am ready to start using a confocal microscope and do some
> immunohistochemistry work.
>
> I have found three methods I would like to try. I would really appreciate it if I
> could get some constructive criticsm on them.
>
> Antibody: Sheep polyclonal anti – caffeine 5mg/ml.
>
> Tracer: Fluorescein thiocarbamyl ethylendiamine (FTC-ED) (30nM/L working
> concentration).
>
> Diluent buffer: Na – phosphate buffer (100mM, pH 7.4) containing 1g/L bovine
> gamma – globulins and 1g/L sodium azide. (Suppliers recommendation) or just
> PBS and BSA is fine.
>
> 1.)	Immunostaining of fixed samples:
>
> Samples: Leaf blade containing caffeine, chemically fixed with formaldehyde,
> dehydrated with EtOH and embedded in L.R. White or high pressure frozen and
> freeze substituted in EtOH and formaldehyde and embedded in L.R. White or
> high pressure frozen, freeze dried and embedded in L.R. White.
>
> Procedure (direct labeling) obtained from light microscopy in biology edited by
> A. J. Lacey:
>
> 1.)	Make sections of +/- 1 – 5µm thick.
> 2.)	Fix samples to slide using water or buffer (PBS) by applying heat (i.e.
> evaporation). Or ‘free float samples’.
> 3.)	Circle sections using a PAP pen.
> 4.)	Place slide in humidity chamber (plastic Petri dish with water soaked
> tissue and slide raised on a plastic bar).
> 5.)	Place 0.5% (or 2%?) BSA , PBS on fixed sample for 10 - 15min.
> 6.)	Rinse in PBS and wipe excess away.
> 7.)	Place antibody on fixed samples in appropriated dilution i.e. 1:100.
> Incubate for 1 hour at room temperature.
> 8.)	Wash fixed sample in 0.5% (or 2%) BSA in PBS.
> 9.)	Wash fixed sample in PBS, 0.01% detergent (Triton x100 or Tween
> 80 in order to reduce the non-specific interaction between the Ig’s and the
> proteins in the section.). Remove excess.
> 10.)	Mark PAP pen marking with a black marker.
> 11.)	Add a small amount of glycerol antifade mountant (n – propylgallate
> or vectashield or 2mg/ml ascorbic acid etc) and add coverslip.
>
> Questions:
> 1.)	What would be a better buffer to use? PBS or sodium phosphate?
> 2.)	Is it necessary to work in sterile conditions i.e. autoclave material
> etc?
> 3.)	Do you think there are unnecessary wash steps i.e. steps 6 and 8? I
> am worried about washing out the antigen.
>
> 2.) Immunostaining of fresh samples:
>
> Sample: Fresh leaf containing caffeine.
>
> Procedure: obtained from a PhD thesis titled Immunological localization of plant
> secondary metabolites by Dr. Louise Brisson:
>
> 1.)	Make hand sections with a sharp razor blade +/- 1mm thick.
> 2.)	Adhere sample to slide using water or buffer (PBS) and mark with a
> PAP pen or ‘free float samples’ and place slide in humidity chamber as
> mentioned in section 1 (step 2 – 4).
> 3.)	Block using 1% w/v gelatin in PBS (or osmoticum) for 15min.
> 4.)	Incubate in antibody as mentioned in 1 (step 7).
> 5.)	Rinse 3 times 3 min using PBS.
> 6.)	Mount as in section 1 (step 10 and 11).
>
> Questions:
>
> 1.)	Should I include a step where the thick sections are incubated in
> buffer and formaldehyde as a fixative?
> 2.)	Instead of using gelatin in step 3 is it ok just to use BSA as in
> section 1.
> 3.)	Is it necessary to include a wash step before incubating with the
> antibody i.e. just PBS?
> 4.)	After incubation should I wash as in section 1 step 8 and 9 i.e. first
> with PBS, BSA (or gelatin) then with PBS with detergent instead of 3 times 3
> min using PBS?
> 5.)	There is no step that includes the use of a detergent? Could this be
> right? How could the antibody cross membranes then?
> 6.)	Should I try this procedure as it is?
>
>
> 3.) Immunostaining of cryofixed/cryoultramicrotome samples.
>
> Sample: Fresh leaf containing caffeine.
>
> Procedure: obtained from a PhD thesis titled Immunological localization of plant
> secondary metabolites by Dr. Louise Brisson:
>
> 1.)	Cut sections as in section 2 step 1.
> 2.)	Embed in 20% w/v gelatin and rapidly freeze in N2 (l) and section
> using a cryo ultramicrotome (+/- 8mm thick). Or use any type of cryo method
> to achieve good sections.
> 3.)	Labeling and fixing to a slide is done as steps 2 – 6 in section 2.
>
> Questions:
>
> 1.)	My questions are the same for section 2.
> 2.)	Is it necessary to include a detergent since the sections are so thin?
> Should the detergent only be included as a wash in order to reduce the non-
> specific interaction between the Ig’s and the proteins in the section.
>
>
> Thank you for reading my methods and any comments are welcomed.
>
> Regards,
>
> Shane Vontelin van Breda.
> MSc. Biochemistry.
> University of Pretoria.
> Department of Biochemistry.
> +27 83 998 6281.
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