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.)
- Prepare Physical Developer Solutions A, B & C
- Nitric acid wash all glassware.
- 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.
- Prepare SPC Solutions. Stir until needed. Prepare other solutions.
- Place sections in 2% NH4OH. Stir on stirring table for 5 min.
- Place in dH2O 2 x 1 min. each.
- Place in the SPC Soln. and cover. Stir gently for 40 min.
- Place in 1% Citric Acid for 3 min.
- Place in 4.99 pH Acetate Buffer Working Solution until ready for Physical Developer.
- Prepare fresh Physical Developer ABC Sol’n. for each batch. Develop 10-15 sections at a time. Use fewer sections if they appear crowded.
- Place in Physical Developer ABC Sol’n. over a light source. The development time is visually assessed.
- Stop development by placing in 4.99 pH Acetate Buffer Working Solution briefly.
- Place in fresh 4.99 pH Acetate Buffer Working Solution. Timing is not critical.
- Place in fresh 4.99 pH Acetate Buffer Working Solution. Timing is not critical.
- Place into fresh dH2O 30 sec.
- Place into 0.5% Sodium Thiosulfate solution for 45 sec.
- Place into fresh dH2O 3 x 2 min. each
- Mount sections.
- Coverslip.
Other links of possible interest:
- The original abstract introducing the Campbell-Switzer method quantitative analysis of staining results
- About the Campbell-Switzer method
- For comparison of CS stain with antibody stains See Schonheit et al
- NSA offers digital, systematic analysis of the stained sections from the Campbell-Switzer stain. For details, refer to our Image Analysis page.
FAQ regarding silver stains:
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.
Yes. There are optimal times for observing synaptic terminals, cell bodies and axons. See our reference section for more information.
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.
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.
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.
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 dH2O | Prepare and mix well. |
| 1.0 g AgNO3 | |
| 100 ml 1% AgNO3 |
| Silver Stain Solution #2: 1% Potassium Carbonate (K2CO3) | |
|---|---|
| 100 ml dH2O | Prepare and mix well. |
| 1.0 g K2CO3 | |
| 100 ml 1% K2CO3 |
| Silver Stain Solution #3: Silver-Pyridine-Carbonate (SPC) Batch Sizes | ||||||
|---|---|---|---|---|---|---|
| 1% AgNO3 | 60 ml | 120 ml | 180 ml | 240 ml | 300 ml | 360 ml |
| Pyridine | 17 ml | 34 ml | 51 ml | 68 ml | 85 ml | 102 ml |
| 1% K2CO3 | 45 ml | 90 ml | 135 ml | 180 ml | 225 ml | 270 ml |
| Total Volume: | 122 ml | 244 ml | 366 ml | 488 ml | 610 ml | 732 ml |
| Silver Stain Solution #4: 2% Ammonium Hydroxide | |
|---|---|
| 98 ml dH2O | Prepare and mix well. |
| 2 ml NH4OH (27-29% concentrate) | |
| 100 ml 2% NH4OH |
| Silver Stain Solution #5: 1% Citric Acid | |
|---|---|
| 100 ml dH20 | Prepare 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 Acid | Prepare 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 dH2O | Prepare 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 dH2O | Prepare and mix well. |
| 2 g Sodium Thiosulfate | |
| 400 ml 0.5% Sodium Thiosulfate |
| Silver Stain Solution #8a: Physical Developer | |
|---|---|
| Solution A | Prepare and mix well. |
| 500 ml dH2O | |
| 25 g Na2CO3 | |
| 500 ml Solution A | |
| Solution B | Prepare and mix. |
| 500 ml dH2O | |
| 1.0 g NH4NO3 | |
| 1.0 g AgNO3 | |
| 5.0 g Tungstosilicic Acid | |
| 500 ml Solution B | |
| Solution C | Prepare 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 Sections | Paraffin Sections | |
| Solution A | 100 ml | 100 ml |
| Solution B | 90 ml | 80 ml |
| Solution C | 10 ml | 20 ml |