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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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