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NeuroScience Associates

Campbell-Switzer Alzheimer Silver Stain

 

Plaques and Tangles: A Controlled and Enhanced Method

Staining Procedure:

(125×65 dishes for up to 30 sxns max & 100×50 dishes for 13 or less sxns.)

  1. Prepare Physical Developer Solutions A, B & C
  2. Nitric acid wash all glassware.
  3. Remove sections from storage containers and place in dH2O 3 x 10 min each. Use baskets to transfer sections through all steps except the SPC and Physical Developer ABC steps.
  4. Prepare SPC Solutions. Stir until needed. Prepare other solutions.
  5. Place sections in 2% NH4OH. Stir on stirring table for 5 min.
  6. Place in dH2O 2 x 1 min. each.
  7. Place in the SPC Soln. and cover. Stir gently for 40 min.
  8. Place in 1% Citric Acid for 3 min.
  9. Place in 4.99 pH Acetate Buffer Working Solution until ready for Physical Developer.
  10. Prepare fresh Physical Developer ABC Sol’n. for each batch. Develop 10-15 sections at a time. Use fewer sections if they appear crowded.
  11. Place in Physical Developer ABC Sol’n. over a light source. The development time is visually assessed.
  12. Stop development by placing in 4.99 pH Acetate Buffer Working Solution briefly.
  13. Place in fresh 4.99 pH Acetate Buffer Working Solution. Timing is not critical.
  14. Place in fresh 4.99 pH Acetate Buffer Working Solution. Timing is not critical.
  15. Place into fresh dH2O 30 sec.
  16. Place into 0.5% Sodium Thiosulfate solution for 45 sec.
  17. Place into fresh dH2O 3 x 2 min. each
  18. Mount sections.
  19. Coverslip.

 

Other links of possible interest:

FAQ regarding silver stains:

Can I execute the degeneration stain in my own lab?

Yes. The foundation protocols are published, but it took NSA years to perfect our own variation and achieve consistent success and high quality in executing these stains.

Is the time between injury/insult and when the animal is sacrificed important when assessing neurodegeneration?

Yes. There are optimal times for observing synaptic terminals, cell bodies and axons. See our reference section for more information.

Are silver stains all the same?

No. There are numerous silver staining protocols, each tailored to reveal specific features. "Silver Stains" are as unique as any other stain in that they are each specifically designed to stain a particular feature. Silver simply refers to the staining agent (Silver) which stains the designated feature black. See: Switzer, R. C. III. Application of Silver Degeneration Stains to Neurotoxicity Testing. Toxicologic Pathology 28: 70-83, 2000.

Is FluoroJade as effective in staining degeneration as the silver degeneration stains?

No. Researchers are reporting that some forms of degeneration are unstained by FluoroJade. Also, as FluoroJade is a fluorescent stain, sections cannot be viewed practically at low magnifications as can be done with the amino cupric silver stain.

Does the Campbell-Switzer stain show the same features as the disintegrative degeneration stain?

No. Each method reveals a different set of features. The Campbell-Switzer method reveals the neuritic plaques and tangles of Alzheimer's pathology. The disintegrative degeneration stain shows the products of neuronal disintegrative degeneration.

How do the plaques revealed by the Campbell-Switzer stain in transgenic mouse models compare with staining using antibodies against different forms of amyloid?

Some antibodies show only a subset of plaques revealed by the Campbell-Switzer method, while others appear to be a one to one match. The Campbell-Switzer stain is unique in its ability to differentiate its staining of immature (diffuse) and mature (congophilic) plaques, allowing researchers a unique observational capability.

 

For further reference, please see: Stains

Solutions:

Silver Stain Solution #1: 1% Silver Nitrate (AgNO3) 
100 ml dH2OPrepare and mix well.
1.0 g AgNO3
100 ml 1% AgNO3
Silver Stain Solution #2: 1% Potassium Carbonate (K2CO3) 
100 ml dH2OPrepare and mix well.
1.0 g K2CO3
100 ml 1% K2CO3
Silver Stain Solution #3: Silver-Pyridine-Carbonate (SPC)

Batch Sizes
      
1% AgNO360 ml120 ml180 ml240 ml300 ml360 ml
Pyridine17 ml34 ml51 ml68 ml85 ml102 ml
1% K2CO345 ml90 ml135 ml180 ml225 ml270 ml
Total Volume:122 ml244 ml366 ml488 ml610 ml732 ml
Silver Stain Solution #4: 2% Ammonium Hydroxide 
98 ml dH2OPrepare and mix well.
2 ml NH4OH (27-29% concentrate)
100 ml 2% NH4OH
Silver Stain Solution #5: 1% Citric Acid 
100 ml dH20Prepare and mix well.
1.0 g Citric Acid
100 ml 1% Citric Acid
Silver Stain Solution #6a: 4.99pH Acetate Buffer Stock 
60 ml 1M Acetic AcidPrepare and mix well.
140 ml 1M Sodium Acetate
200 ml 4.99 pH Acetate Buffer Stock
Silver Stain Solution #6b: 4.99pH Acetate Buffer Working Solution 
238 ml dH2OPrepare and mix well.
12 ml 4.99 pH Buffer
250 ml 4.99 phH Acetate Buffer Working Solution
Silver Stain Solution #7: 0.5% Sodium Thiosulfate 
400 ml dH2OPrepare and mix well.
2 g Sodium Thiosulfate
400 ml 0.5% Sodium Thiosulfate
Silver Stain Solution #8a: Physical Developer 
Solution APrepare and mix well.
500 ml dH2O
25 g Na2CO3
500 ml Solution A
Solution BPrepare and mix.
500 ml dH2O
1.0 g NH4NO3
1.0 g AgNO3
5.0 g Tungstosilicic Acid
500 ml Solution B
Solution CPrepare and mix well.
250 ml dH2O
0.5 g NH4NO3
0.5 g AgNO3
2.5 g Tungstosilicic Acid
1.75 ml 37% Formaldehyde
250 ml Solution C
Silver Stain Solution #8b: Physical Developer ABC  
Free-Floating SectionsParaffin Sections
Solution A100 ml100 ml
Solution B90 ml80 ml
Solution C10 ml20 ml
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For clients with projects in process: NSA will communicate with you prior to shipping materials to ensure you are able to accept deliveries. If you are unable to receive deliveries due to the current global situation, NSA will hold shipments at our facility until your delivery system returns to normal. If you have not added digital imaging to your order but need it to expedite or continue your research, contact us right away so we can work together to meet your needs.

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