BIOE 202 Section AB1

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BIOE 202Section AB1

• Lab 12
• Calcium StainingLysate Preparation

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Lab 12 Overview

• Review protocols and information
• Staining for alkaline phosphatase and calcium
• Osteoblast Lysates
• Photograph Neurons

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A. Staining Lab manual p. 112

• Stain 4 wells total (1wk undiff, 1,2,3 wk diff)
• Goggles and gloves are required
• Use your PBS
• Take your plates to the hood to add reagents, but
  rinses can be done at your bench

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A. Staining Lab manual p. 112

• After step four, cover staining wells with
  parafilm to start lysates
• There are separate waste containers for each
  reagent. Please use the correct one.
• Anything touched by silver nitrate is hazardous
  and must go in special bags

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B. Osteoblast LysatesLab manual p. 113

• Keep everything on ice
• Start with undifferentiated well, then 1, 2, and
  3 weeks
• Week 3 might be hard to scrape offbut you can
  push hard without worrying

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C. Photograph NeuronsLab manual p. 114

• View neurons and write down observations
• Take a photo
• Bleach plates when done
• Coverslips go in sharps container
• Plates go in trash

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Return Day

• No Return Day!

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Reminders

• Online quizzes only one more
• Final exam during class April 30th
• Third lab report due May 3rd

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Order of Events

• All groups start with part A
• Start part B after step 4 of A
• Movie during waits
• Miracle Cell

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Online Quiz Results

• Osteoblasts secrete alkaline phosphatase to
  increase the local concentration of phosphate
  ions
• Osteoblasts secrete osteocalcin to increase the
  local concentration of calcium ions
• Collagen is the main component of osteoid
• In vivo most unmineralized bone osteoid is found
  just underneath the periosteum
• Every 3 days new osteoblasts push older
  osteoblasts toward mineralized bone and encase
  them
• The D1 cells are performing intramembranous
  ossification when differentiating
• In vivo bone formation absolutely requires
  vascularization to have a source of calcium ions,
  but we are forming bone in vitro, which does not
  require vascularization
• Von Kossa staining involves fixing with NFB,
  staining for ALP with red violet, and staining
  for mineralized bone with silver nitrate
• The pipet for silver nitrate requires special
  disposal