Histochemistry via lead precipitation

 

This enzyme histochemical technique demonstrates NTPDase2 activity (which is an extracellular ATPase)

 

Do not want to use anything with phosphate in it...phosphate will be provided with ATP or ADP...therefore, the buffer used is tris-maleate

 

Technique outlined in the previous publication:

Title:  Localization of Nucleoside Triphosphate Diphosphohydrolase-1 (NTPDase1) and NTPDase2 in Pancreas and Salivary Gland

Authors:  Kittel A, Pelletier J, Bifonnesse F, Guckelberger O, Kordas K, Braun N, Robson SC, Sevigny J

Journal:  Journal of Histochemistry & Cytochemistry  Volume 52(7): 861-871 (2004)

 

                                                                                                                                               

 

Perfusion and fixation

 

After animal is anesthetized, clear blood with 0.9% NaCL and perfusion fix with following (last page gives tris-maleate recipe):

 

            PFA                             final concentration 2%

            glutaraldehyde             final concentration 0.2%

            CaCl2                           final concentration 2mM

            made in 0.1M tris-maleate buffer

 

Remove the tongue from lower jaw and postfix 3 x 20 minutes in the above fixative, shaking at room temperature.

Place tongue in 20% sucrose made in 0.1M tris-maleate buffer overnight at 4C.

 

Embed taste papillae in OCT, cut on cryostat at 14-16 um, collect on superfrost slides, and store at –20C overnight.

 

 

Enzyme Histochemistry

 

Remove slides to be processed from freezer, let come to room temperature.  Dip in dH2O and dry on hotplate.  Remove from hotplate into 70mM tris-maleate and wash 2 x 10 minutes.

 

 

 

 

 

 

Make the following ‘stock’:  (amounts on the left need to be added to the total volume of 10mL to yield the final concentration on the right: these amounts are based on MW of the reagents listed on last page)

 

per 10mL of 70mM tris-maleate (this solution should be milky-white in appearance)

 

6.624mg          Pb(NO3)2                   2mM

2.408mg          levamisole                   1mM    (inhibits alkaline phosphatases)

5.847mg          ouabain                        1mM    (inhibits Na,K-ATPase)

3.728mg          KCl                             5mM

212.6ug           αβ-methylene ADP      50uM   (inhibits 5’-nucleotidases)

2.22mg CaCl2 (anhydrous)            2mM

 

To the above ‘stock’ will be added different substrates.

(The experiment is to use ATP in the presence of lead nitrate and all of the other ATPase inhibitors.  The controls are to use ADP instead of ATP and no substrate at all.)

 

Determine how much volume is needed with ATP (calculate 200ul/slide)

The final concentration of ATP needs to be 1mM

Example:         6 ml of stock

= 3.5mg of ATP

 

Determine how much volume is needed with ADP

The final concentration of ADP needs to be 1mM

Example:         3 ml of stock

                                    =1.28mg of ADP

 

Determine how much volume is needed without substrate as use as is from the stock.

 

 

Apply solutions to the slides, coverslipe with parafilm, and let incubate for 30 minutes at room temperature.

 

Drain slides and wash 3 x 10 minutes 70mM tris-maleate

 

Incubate slides with 1% (NH4)S (ammonium sulfide) for 1 minute (this converts the lead precipitate to PbS)

 

Drain slides and wash 2 x 10 dH2O

 

Slides can be coverslipped with fluormount G or processed further using a nuclear stain like thionin (very pretty) if desired.  If counterstain used, dehydrate through ethanol to zylene and coverslip with Permount.

 

 

 

Tris-maleate buffer

 

Stock A (0.2M)

24.2g   tris-hydroxymethyl aminomethane

23.2g   maleic acid

add to 1 liter water

 

Stock B (0.2N)

1.6g NaOH

add to 200mL water

 

To make 0.1M tris-maleate with pH 7.4

per 100mL

 25mL stock A

 27mL stock B

 48mL water

 

To make 70mM tris-maleate

            per liter

            700mL 0.1M tris-maleate

            300mL water

 

 

Reagents

 

tris hydroxymethyl aminomethane                               Fisher              CAS 77-86-1

maleic acid                                                                  Sigma              M0375

NaOH                                                                          Sigma              S5881

CaCl2 (anhydrous)                                                                                            Fisher             

Pb(NO3)2                                                                   Sigma              L6258

levamisole                                                                   Sigma              L9756

ouabain                                                                        Sigma              O3125

KCl                                                                             Sigma              P9541                         

αβ methylene ADP                                                      Sigma              M3763

ADP                                                                            Sigma              A5285

ATP                                                                             Sigma              A8937