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FFPE 肾组织 PAS 染色

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This protocol adopts the FFPE branch of the protocols.io PAS staining protocol for human kidney tissue v3. Paraffin sections are dewaxed in xylene, rehydrated through graded alcohols, oxidized with periodic acid, stained with Schiff reagent, counterstained with hematoxylin, dehydrated, cleared and coverslipped to demonstrate glomerular basement membranes, mesangial matrix and glycoproteins. The record describes the staining procedure, not executed disease-model results.

Scope and method

Sample：FFPE kidney sections

染色

Reagents and materials

Procedure

  1. Start with FFPE here:

    Allow PAS “kit” to come to room temperature on the bench.

  2. Start with FFPE here:

    For paraffin sections, deparaffinize in xylene, two changes, 00:03:00 each.

  3. Start with FFPE here:

    Hydrate through graded alcohols, 00:01:00 each: 100%, 100%, 95%, 70%, water

  4. Start with FFPE here:

    Rinse well in distilled water by holding finger over slides, pouring water into sink and adding water. Do this 5 times.

  5. Start Frozen samples here:

    Place in 0.5% Periodic acid for 00:10:00 (to oxidize).

  6. Start Frozen samples here:

    Rinse well in distilled water (follow Step 4).

  7. Start Frozen samples here:

    Pour Schiff’s reagent into Coplin jar containing slides. Allow to sit 15-30 minutes at room temperature (kidney samples ~30 minutes).

  8. Start Frozen samples here:

    Rinse in running warm (~40 °C) distilled or tap water (follow Step 4). The tissue should appear pink after rinsing with warm water.

  9. Start Frozen samples here:

    Place slides in movable gray slide holder.

  10. Start Frozen samples here:

    Counterstain in Hematoxylin (staining line) for 00:01:00.

  11. Start Frozen samples here:

    Rinse well in distilled water, starting with blue container next to hematoxylin (follow Step 4).

  12. Start Frozen samples here:

    Quickly dip slides into “Bluing” agent (0.5% ammonium hydroxide).

  13. Start Frozen samples here:

    Rinse 1 minute in distilled water until pink color is visible (follow Step 4).

  14. Start Frozen samples here:

    Dehydrate through graded alcohols, 10 short dips in each: 95%, 95%, 100%, 100% If the last 100% ethanol rinse is colored by residual stain, additional washing in new 100% ethanol solution should be performed.

  15. Start Frozen samples here:

    Fix in 2 rounds of xylenes, 00:01:00 each (in the hood).

  16. Start Frozen samples here:

    Coverslip slides: - Place coverslip on paper towel - Add 2-3 drops of cytoseal to edge (depending on size) - Dip slide into xylenes, take out and roll the xylene lengthwise on slide - Line up slide and cover slip and slowly place the slide on the coverslip - If needed, use dissecting tool to remove bubbles

  17. Start Frozen samples here:

    Image slides using a Leica brightfield scanner, AxioScan Z1 slide scanner (or equivalent) at 20x (~1 µm spatial resolution) and save as.tiff or.jpg file.

Quality control and readouts

脱蜡与复水梯度按源方案执行；Schiff 试剂避光并在有效期内使用；高碘酸氧化时间与苏木素复染时间保持一致；每批染色设阳性对照组织并检查核与基底膜对比；封片后避免气泡和干片。

肾小球基底膜、系膜基质和糖蛋白呈 PAS 阳性（品红/紫红色），细胞核呈蓝色；按研究设计进行肾小球系膜扩张或病变范围判读。

Study design
FFPE 肾组织切片的 PAS 染色流程，按源方案 FFPE 分支执行脱蜡、复水、氧化、Schiff 显色、苏木素复染、脱水透明和封片。具体切片厚度、视野与定量方法按研究设计确定。

References

  1. Elizabeth Neumann, Jamie Allen, Angela R.S. Kruse, Jennifer Harvey, Maya Brewer, Carrie Romer, Mark De Caestecker, Jeff Spraggins. PAS Staining of Fresh Frozen or Paraffin Embedded Human Kidney Tissue. protocols.io v3, 2023. https://doi.org/10.17504/protocols.io.x54v98nkzl3e/v3

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